<%BANNER%>

Characterization of Actin-Based Motility on Modified Surfaces for In Vitro Applications in Nanodevices

Permanent Link: http://ufdc.ufl.edu/UFE0021656/00001

Material Information

Title: Characterization of Actin-Based Motility on Modified Surfaces for In Vitro Applications in Nanodevices
Physical Description: 1 online resource (154 p.)
Language: english
Creator: Interliggi, Kimberly A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: biomotors, bionanodevices, elongation, listeria, lithography, myosin, nanoparticles, tirf
Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The cytoskeletal protein actin generates forces for various processes by polymerizing into filaments. In vivo, actin works with the motor protein myosin to produce muscle contractions and with proteins acting as end-tracking motors responsible for cell and bacterial motility, such as the motility of Listeria monocytogenes. End-tracking proteins bind the polymerizing end of an actin filament to a motile surface, creating persistent attachment during filament elongation. Both types of motors use the energy from ATP hydrolysis and can be exploited in vitro in bionanodevices, which require forces to transport objects on a micro- or nano-scale, possibly against flow or diffusion gradients. Our study has focused on the guidance of single-filament elongation and filament bundles (rocket tails) to orient elongation in vitro. Microcontact printing, a technique that stamps protein patterns onto glass surfaces through adsorption, was used to create filament-binding tracks of modified myosin (void of its motor activity), which successfully bound and guided single actin filament elongation in a manner dependent on track width and surface conditions. These results confirm the capability of this method to be used for the motility of objects attached to single actin filaments and for the creation of immobilized tracks of actin filaments for myosin mediated transport. Simulations were used to characterize the system further and have the ability to help make predictions for other types of filaments and systems. Modified myosin surfaces also confined actin rocket tails attached to particles and bacteria, reducing the Brownian motion of the motile objects. Channels formed through photolithographic techniques on glass surfaces were used to attempt to guide these particles. Single actin filaments attached to smaller particles were also characterized to determine the potential for single-filament propulsion in nanodevices. We conclude that actin filament-binding proteins can be applied to surfaces using adsorption and microcontact printing and that this technique is effective in binding and guiding filaments in various systems, including single and bundled filaments. We predict this technique can be applied to other systems undergoing actin based motility, making it a versatile method for bionanotechnology.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kimberly A Interliggi.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Dickinson, Richard B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021656:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021656/00001

Material Information

Title: Characterization of Actin-Based Motility on Modified Surfaces for In Vitro Applications in Nanodevices
Physical Description: 1 online resource (154 p.)
Language: english
Creator: Interliggi, Kimberly A
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: biomotors, bionanodevices, elongation, listeria, lithography, myosin, nanoparticles, tirf
Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The cytoskeletal protein actin generates forces for various processes by polymerizing into filaments. In vivo, actin works with the motor protein myosin to produce muscle contractions and with proteins acting as end-tracking motors responsible for cell and bacterial motility, such as the motility of Listeria monocytogenes. End-tracking proteins bind the polymerizing end of an actin filament to a motile surface, creating persistent attachment during filament elongation. Both types of motors use the energy from ATP hydrolysis and can be exploited in vitro in bionanodevices, which require forces to transport objects on a micro- or nano-scale, possibly against flow or diffusion gradients. Our study has focused on the guidance of single-filament elongation and filament bundles (rocket tails) to orient elongation in vitro. Microcontact printing, a technique that stamps protein patterns onto glass surfaces through adsorption, was used to create filament-binding tracks of modified myosin (void of its motor activity), which successfully bound and guided single actin filament elongation in a manner dependent on track width and surface conditions. These results confirm the capability of this method to be used for the motility of objects attached to single actin filaments and for the creation of immobilized tracks of actin filaments for myosin mediated transport. Simulations were used to characterize the system further and have the ability to help make predictions for other types of filaments and systems. Modified myosin surfaces also confined actin rocket tails attached to particles and bacteria, reducing the Brownian motion of the motile objects. Channels formed through photolithographic techniques on glass surfaces were used to attempt to guide these particles. Single actin filaments attached to smaller particles were also characterized to determine the potential for single-filament propulsion in nanodevices. We conclude that actin filament-binding proteins can be applied to surfaces using adsorption and microcontact printing and that this technique is effective in binding and guiding filaments in various systems, including single and bundled filaments. We predict this technique can be applied to other systems undergoing actin based motility, making it a versatile method for bionanotechnology.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kimberly A Interliggi.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Dickinson, Richard B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021656:00001


This item has the following downloads:


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101118_AAAABQ INGEST_TIME 2010-11-18T14:49:31Z PACKAGE UFE0021656_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 6760 DFID F20101118_AABBXB ORIGIN DEPOSITOR PATH interliggi_k_Page_039thm.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
381471c73b9a2058f1cc061faed73ce2
SHA-1
941e66d1f5bfa9ec4ea9ba8c0e7d6ffe789e39d4
53678 F20101118_AABCBS interliggi_k_Page_120.pro
02a61f4dcc951882e232f942467a5722
dfe10be1a01fd20a567bd22ad0cd23b94028c40c
30965 F20101118_AABBWM interliggi_k_Page_007.pro
18cca43d3340862646095cb0575bbb1e
19dd9b3bc400c414d43709e989abaaac32739ad3
51977 F20101118_AABBVY interliggi_k_Page_128.jpg
ad15a438d2f248ea33ed95e4c2f01feb
742c21314a100e742e46149d6d3f63a158c55830
911111 F20101118_AABCCH interliggi_k_Page_116.jp2
adaf4926c293686ba715420f426c7bd0
f8f53a4d8dc70cb366a6ffc46b741a091d57e52b
1051973 F20101118_AABCBT interliggi_k_Page_072.jp2
a4d96b189054c2201fa4677c56967163
3eb12e0dc87a4992f10cc63d00d94ed406e13ec9
453079 F20101118_AABBWN interliggi_k_Page_103.jp2
c627009d5d84d8bd41cbe28f634b15ca
d94ebe537452edcfe74ef2caa826ff618b11957c
25271604 F20101118_AABBVZ interliggi_k_Page_069.tif
2460673553e1e00cceadc78a2a2c44dc
29b869330305a892f8e9afff8891e0da649c3302
4108 F20101118_AABCCI interliggi_k_Page_057thm.jpg
2ee55936196e3e895cdd82154968cb3a
958f2434d7d8ed324f42851e81371d25017002ac
27749 F20101118_AABBXC interliggi_k_Page_128.pro
f33ea195aef90fda29fca92503d40570
953f434bb32c460b3342a9d47eb24ea69179525c
F20101118_AABCBU interliggi_k_Page_034.tif
058069e47f882d8e8658d5867793e8ed
82c4a3e56cf627a13f8cfbdb200a2ca7281e7f3b
F20101118_AABBWO interliggi_k_Page_079.tif
b97db1180a9ffae1713577242dc788f3
6b0c461275404bae33ff7b39461f2ed46fc7d4d2
2176 F20101118_AABCCJ interliggi_k_Page_137.txt
2583a391cb74e5f404531e7937380c63
4354d0753689ae7cefa39760413649ab92f701b1
1051965 F20101118_AABBXD interliggi_k_Page_148.jp2
6619c37dce5fe1f3b2326b423a5fcf26
996b43293b6eebff0afbfb14b9d4ffa0530be336
26453 F20101118_AABCBV interliggi_k_Page_114.QC.jpg
f77b69f1412db43c00dcdd14d97cce5f
69d46ff982d56d611e6d05d8c201e63cd973c923
16958 F20101118_AABBWP interliggi_k_Page_103.pro
45b0df31fc4dc21107f03844b2719fb3
d81ec155e89beabec091ecf0de108090b10a78da
6346 F20101118_AABCCK interliggi_k_Page_032thm.jpg
65c5243e5770560f7945e93eae29178b
93256a1b59cf75a04d3e4d78ebaf5c7e5c27c7af
6562 F20101118_AABBXE interliggi_k_Page_034thm.jpg
53f9c9313617b4e59b95e83722cd5336
be3ccf8dff47ee8a0f319cd2b25b42e62a017984
77881 F20101118_AABCBW interliggi_k_Page_030.jpg
8156eee1e01c94c7e3f5ced6e05e7a1d
8e426502f280ff16597a2b700ba94cc5484a6d3f
2964 F20101118_AABBWQ interliggi_k_Page_011thm.jpg
f040423a3c39340ee47e1499d2c0df25
847b86289606b22ff1f1f282820cf1e59d029816
90412 F20101118_AABCCL interliggi_k_Page_134.jpg
c8ef7ad7b5a0c6061fcacf03acd6dbff
dba90a2b937e88345fe65d89b339a77f307bf9a4
53375 F20101118_AABBXF interliggi_k_Page_141.pro
b059d849adc7c7b25f1352fb491a8058
85a6ac98dac868ead31a02ad31149a873021cd00
44623 F20101118_AABCBX interliggi_k_Page_126.jpg
d51b21e25b1b2b29b5d135859ce7a7ee
6922c36df519a36fc640ffc3fc587fd7e2f91dcf
6258 F20101118_AABBWR interliggi_k_Page_028thm.jpg
3d7f21956cf4e568b2d1ce1697727426
392f70d2d2eea48f23c629052f0d39f0a07a0006
65757 F20101118_AABCDA interliggi_k_Page_105.jpg
fedc9e82be4e35bdb216f17d338d3220
2245714a7c03508265d2f73b7615c41212a9b62a
26436 F20101118_AABBXG interliggi_k_Page_029.QC.jpg
a6e588b25e01749bdf96724577d523ca
3e481b7f801ad0717f3675cdf59b9be019e3bab3
18587 F20101118_AABCBY interliggi_k_Page_047.pro
b2d2f67fbf71d1b490fbf9156cbedc41
67c744482c16cee62e477902ee9f5c1dd4992d32
48686 F20101118_AABBWS interliggi_k_Page_019.pro
df038ff16a4b7e5a23e8618e9754a123
382f5accbc2472e3d167bd7be068c31d9dceb87d
30651 F20101118_AABCDB interliggi_k_Page_123.jpg
3748d64213be31d8bd6795eb18b97305
6ed37c71e39edc8ab3b70f90f475634c86619629
5806 F20101118_AABCCM interliggi_k_Page_019thm.jpg
4e9d2e3d3bf8f98e832eb43bf8f73c01
87e8a890a696e05364b6c919fc9b35d333bb0d51
801 F20101118_AABBXH interliggi_k_Page_104.txt
883e26c5994ab1dec0528f922293e149
95ab6df9c579814225d13653656f2a75c4cbdd75
7135 F20101118_AABCBZ interliggi_k_Page_001.QC.jpg
9d3d4772c5229424ba02cd0654c9cf2d
559b96504b9855753e9cbbee103bea842973de62
2205 F20101118_AABBWT interliggi_k_Page_038.txt
93fc70c4b47adbcb5940e3b3d31b82b3
04d776985308340500ef6a87f50527dbc858540f
1194 F20101118_AABCDC interliggi_k_Page_079.txt
1ac37cb1ba84ac2915c3c8d37de105ff
c003c6d9a245e89cb8dbda85ff927f2a772b15e6
5302 F20101118_AABCCN interliggi_k_Page_083thm.jpg
e2f209949cc5cf30eb24d74a2f65cd2e
b62634f61d83806ff486df80eba2ab2e99ae1ce3
16724 F20101118_AABBXI interliggi_k_Page_131.pro
315995bb9169fd7af0e98d39122896e1
eb8290fd3ac2b3c7cf81d2809c4de65df3a2dfb4
53797 F20101118_AABBWU interliggi_k_Page_082.jpg
6307fbf0bc0539ab2558b78155c87fda
4dade7d19f5ba6abaa3143035fefc64586e3a0cf
52770 F20101118_AABCDD interliggi_k_Page_095.pro
bbf8eed36253c3c0aba0ce5ea40d070c
819862a0f4a1679eb64b420f0db45f88b96cc4eb
10753 F20101118_AABCCO interliggi_k_Page_081.pro
edc1296c9a9f71a12483a75ad2ecaf13
4e5366639380f9b8b631ac5688414ed93b0e8635
54812 F20101118_AABBXJ interliggi_k_Page_134.pro
dee6a22ed7c8665d26f77dc4edf17cd0
2999584327da2962348b8434c75a611f473a7c1e
1435 F20101118_AABBWV interliggi_k_Page_050.txt
4705872b924750bea15fb14950d7dc81
74bc4581ac3074227feccd994e310136fd88dd44
1118 F20101118_AABCDE interliggi_k_Page_002.QC.jpg
181cb69d1ccc8faa24bac0afffb6170a
8db464689f39f25df597d68f26dffa991417a9ea
716874 F20101118_AABCCP interliggi_k_Page_046.jp2
35c990d4c6e8c951bd522cd8b9bc8395
56dff60a62ffbc3777b334c059a37dfcd5c12393
F20101118_AABBXK interliggi_k_Page_090.tif
19db91a33029f0f02c0f4d18595b70ed
bbd40fcd857e3a7b51c1a7be99525b6bdbf6e91a
33632 F20101118_AABBWW interliggi_k_Page_056.pro
96f0d4d526ae687252399f8b7f33a2d0
8fd3e2f830145d2785a51afc3d90056c0a1fc174
85567 F20101118_AABCDF interliggi_k_Page_113.jpg
9e8221ad650254ec971d4c46ac931973
f04c03ba6dfb1c8e7d725e689f89e26f5a4ad7e0
74103 F20101118_AABCCQ interliggi_k_Page_141.jpg
066846b5c241fa2897cf21c0c4eb5595
ff7a831b828d1fa20e6bae852ce57b7c594795e5
1011 F20101118_AABBXL interliggi_k_Page_061.txt
ccbc83da345a123716717c6a25fb1bd8
c014e0504ac7d501c8ec8945d9e1f48756d2b56e
27457 F20101118_AABBWX interliggi_k_Page_075.pro
3baa6ab13904b2c2ab51c043449dcd76
df6c4893a5649f1498cdd1cad4d0b33a9af00200
12269 F20101118_AABCDG interliggi_k_Page_060.QC.jpg
1f28c53ef82e9442eb91c20ff7ef3c2b
4708d57b30c6f350a1354df92fc352f50f96565a
1352 F20101118_AABBYA interliggi_k_Page_109.txt
a55897952dd129fca651a927d2e5160c
219dcbe93211ca98f2cab7e67489d939e7b8b255
4003 F20101118_AABCCR interliggi_k_Page_128thm.jpg
616b5471e0d5181016f5fc2aa6aedca4
7847ac878bf6bc10ac06f9adf09cca933f6f638d
1248 F20101118_AABBXM interliggi_k_Page_107.txt
5d37d992f722740ed0739bd4a7bfd186
dbd5b024a237327467670af695c41d0fe5c43dd1
1051966 F20101118_AABBWY interliggi_k_Page_140.jp2
87a4a745d03eb2425410d7f2a53aad5a
0fa7828dc7c0fade466e7294a3ec67da09bdbc42
1051956 F20101118_AABCDH interliggi_k_Page_114.jp2
5712ae521c01b27d2214fa419802ffeb
bd189992be943d90d8e50eccc4a1c440f67243d7
89493 F20101118_AABBYB interliggi_k_Page_087.jpg
519cfb87301c64a72214080bd88c7027
79522078826ee04aba879214e66524814abe69d1
2766 F20101118_AABCCS interliggi_k_Page_142.txt
f75d9fb6322da00d15301f9706f35016
8a038957cb714fcc36df2f307860257c9720c738
25271 F20101118_AABBXN interliggi_k_Page_073.QC.jpg
c005414c949b814ed35bac47e4b166ca
008ee6bcbf0d0ad8b20079b52003d661c897071b
4373 F20101118_AABBWZ interliggi_k_Page_142thm.jpg
a818d481ce22acb7d92ace55dff3044f
be42a11da915d030d6166542f703bd9ecc3d70a6
5721 F20101118_AABCDI interliggi_k_Page_020thm.jpg
ec19f8e5021cedc591638be4f792d650
0eeefbb176c8f68e4acdfc50a1fb00035c932b40
6722 F20101118_AABBYC interliggi_k_Page_026thm.jpg
5a415ee21b9e94a40f2a2fc389b840dd
aefff46897017651aad4e19b92a435a3e8073ac9
301871 F20101118_AABCCT interliggi_k_Page_154.jp2
0d92405fcd0df01ac0ab9bb3ec3aa8dc
c13037f3fdad24423418b47eeecb3fa632be73b4
6572 F20101118_AABBXO interliggi_k_Page_096thm.jpg
38842d4abcb8bc08dcc482c2501c23ed
38e03224574a8809321cf78d108358f3fdb6a43e
77038 F20101118_AABCDJ interliggi_k_Page_040.jpg
40e952f84e43324df74aca2cbf1d94d1
b6e5309109511f008915c140c7b230af5841cb93
26374 F20101118_AABCCU interliggi_k_Page_067.QC.jpg
8a64d304ed691a79951a2710f3ed0cf1
35f86b615fc24004f674c62ee08e095a1b4cf56f
2018 F20101118_AABBXP interliggi_k_Page_114.txt
7375c3d828fd10d80d4a1269f6502ee1
9ea452513fc1a067a52018f876d167041df25b66
27720 F20101118_AABCDK interliggi_k_Page_094.QC.jpg
ca295a5e5d10d7f4d520671497a5202b
7528133cd62add496890fe04751c5631b092ed53
F20101118_AABBYD interliggi_k_Page_116.tif
8762614a43022586fb7aaee42387963a
b469e5e73f7a47a6efd7be7a2b7939394ad127dd
25663 F20101118_AABCCV interliggi_k_Page_008.QC.jpg
41576ccc5019dcf3a0edbd7d21dc5169
0aa869dedbcfd12bb9e35ea7bada9719fc77115e
807226 F20101118_AABBXQ interliggi_k_Page_132.jp2
9e0e2a4ae459359500ea94afa4f1744e
9509da4d68d24497ddb838d619839e7535c74d4c
28095 F20101118_AABCDL interliggi_k_Page_135.QC.jpg
92a46b0337ed21bda4265d646eff3365
ea620c4ccd88e5bcef17aea1718206d06730d132
F20101118_AABBYE interliggi_k_Page_146.tif
a56206bd24bcc81e49ac61c566ae9b4f
a8e5dd5f09d3bf8b63469723e30f46bf1c3afb1b
13022 F20101118_AABCCW interliggi_k_Page_108.QC.jpg
58f0c5e200658e814e39e1673e3bbeeb
2b90988997565d55b31b7c38a6de9ec35fa90f43
F20101118_AABBXR interliggi_k_Page_038.tif
953231f1759a1f348cef8bc089fea587
1f56b506f0a8337063f1aa4f83146a61a91bd644
1051972 F20101118_AABCEA interliggi_k_Page_143.jp2
95313b416e23cb847f954961b2b0978e
73c30f5eae55bb525fba8b7faa33a676f89f252e
57323 F20101118_AABCDM interliggi_k_Page_138.pro
be89e55a86936a5c6c3abaf4b498f4a7
9d5c187804a1eafe8ed133cf542fb5125065de66
F20101118_AABBYF interliggi_k_Page_145.tif
6972401faf46496341c35d34a17ea43a
776788cf2cc7ec3845c788c38dcf61933f39eae0
2463 F20101118_AABCCX interliggi_k_Page_076thm.jpg
b34fe0080d769d1a8e9d782abd9a8f06
5e375993ee89292ba1a20bd52ec93ba6743e1249
87811 F20101118_AABBXS interliggi_k_Page_022.jpg
08b043fc3b535ed2bbda7fe1bdf6ae70
abbe8d3b99cd221ec42bc64f9dbd03bc9c41439d
2468 F20101118_AABCEB interliggi_k_Page_145.txt
d272030369103a9128ee4216a49a30d1
00415d11a4d6093628d9ccc7cc738e6a888665a5
720893 F20101118_AABBYG interliggi_k_Page_064.jp2
9aba4803dd89894a905e5c789cc0403f
e2946a7bbfe55f037668ce739cf0cda82ac9d6c5
7165 F20101118_AABCCY interliggi_k_Page_149thm.jpg
c7ec3196352051e9e35decbda2c90ef9
0a4f8d05ea1c79d2343156e0afe9e06d266c95a3
88426 F20101118_AABBXT interliggi_k_Page_026.jpg
8646ba8143a736d458da4ae7d9ac81c7
4c0e12f905226d6b224262450dea41fcf8433d4b
19279 F20101118_AABCEC interliggi_k_Page_105.QC.jpg
0e218f33a213f4ef86deeed8ef139627
90de0857b1b47bf5a8fe110d87ca566fd54c39ab
4190 F20101118_AABCDN interliggi_k_Page_143thm.jpg
085542cf3b04366e89d203013041aac9
53dd647ee949f7adce235d51f02e3ce93c2173a9
3297 F20101118_AABBYH interliggi_k_Page_132thm.jpg
f6b7606529846585311c4000cf191efd
85dc1f71f9c27ec6447b73be7ccecaade0db098a
1910 F20101118_AABCCZ interliggi_k_Page_037.txt
a684a11fa08066f448d47c046f6a9c85
3ef0e2b7cd23e224d2cfa647cdcdb4c3f7f86fd7
744 F20101118_AABBXU interliggi_k_Page_077.txt
dac2307d998a2565a484944bfbccb363
ec8ae85e9e02d65be16b88070cec824a909a4e06
88511 F20101118_AABCED interliggi_k_Page_120.jpg
b3085c0a7d8650a193773546b96ca2bc
4b55b6ac16fe89d21a1a6651ff6579fdf185447a
92537 F20101118_AABCDO interliggi_k_Page_097.jpg
cdf8ed91df5af154564ae86610181905
70b33feeecf1f8de69eb31d70ec90394838058e0
6946 F20101118_AABBYI interliggi_k_Page_138thm.jpg
0c044964bbf52be2c70e68e20888aa80
2749dec4afb493d86ee2314d14c6fdd41bcdf4dd
11089 F20101118_AABBXV interliggi_k_Page_011.QC.jpg
6e219b9bcd298fc01180361c2f237b7c
21f48efae36be91ce15168bccc80c0cf29dbc0eb
12485 F20101118_AABCEE interliggi_k_Page_126.QC.jpg
65e3737dd4bcf8e89b3cc530e459ee9f
33a9dcfc175255bc6232de8e708d94733f9b78c2
7930 F20101118_AABCDP interliggi_k_Page_139.jpg
7df5c9faef68cc5f4ae5915656e2317a
079321e312c2b42ecd8d22288d828517c6d96660
6594 F20101118_AABBYJ interliggi_k_Page_136thm.jpg
26dc6d9618a0a2a86acdae3c36d8f711
2fcefb7d9eef92323fab7c1253af898378d0b9fe
1051975 F20101118_AABBXW interliggi_k_Page_153.jp2
fb7c468747a8a41caf8bb32c68107d5a
0c91a29c4362479e68943dbaa61dd4d1994dbe84
15697 F20101118_AABCEF interliggi_k_Page_054.pro
316b4bb775c119ee0d3a4de5dcee6cf8
a714d5ddd2c8c1596e01b2024c08d1d14b1264a1
F20101118_AABCDQ interliggi_k_Page_142.tif
591bd3237dc18249d0d10c92ca72617a
35185939b8cd616ff9f24894317f8fc517990910
532 F20101118_AABBYK interliggi_k_Page_125.txt
92f2607ef5e05aedcf56f6d34d2d58fb
a90f164b157246766848fccdff5f039eb2795b40
779302 F20101118_AABBXX interliggi_k_Page_060.jp2
ee33d5d200363fb5156e430fea03e8f5
d2389556e47c051d43450a01156c8a0cbe2b8307
5972 F20101118_AABCEG interliggi_k_Page_050thm.jpg
a77aff8b88d855c756255c16f6ea884c
f5778b82ab9435aedc58eff464c6995dbfe6f841
F20101118_AABBZA interliggi_k_Page_106.tif
a98f2b5229cec8361315c75c9444b7e9
19f54b2ad0e9fc4604fc8f441b91a30a474728bb
17804 F20101118_AABCDR interliggi_k_Page_083.QC.jpg
8ca22070b331ddd0ddf921eea693c1a7
18342f5fe629cd62ba78b9351ddae6f91c4c6403
5354 F20101118_AABBYL interliggi_k_Page_140thm.jpg
2420ee0a8cacd7d879c49a9258c7e8a1
2c40ad94a3abceae03817d1a88e18e76d649a58d
979 F20101118_AABBXY interliggi_k_Page_080.txt
7943f7049c22fc347e815b5619fa7a6c
11e35d66f8781c1618282afd07d9092a0dbbd355
52623 F20101118_AABCEH interliggi_k_Page_089.pro
62f6e2f9f36606d22637f5433e181b3b
a049f17502cba7dfb85f96d41773e2f5367d5d64
2696 F20101118_AABBZB interliggi_k_Page_146.txt
7ce0f99cc89d31c2db38d6063a40c5b1
2279ba6f95e5178e0c213111f1d00ba500509d99
2320 F20101118_AABCDS interliggi_k_Page_057.txt
e1a14845dcecf43665006903b95e8ea8
fd526183e7c897adb390dfd64a3682df94433b51
28531 F20101118_AABBYM interliggi_k_Page_087.QC.jpg
73b973ca26b14cd59cc8bfd928348ef1
b91a41f562810a2f72f32aabb697e95a4a94ed6b
31608 F20101118_AABBXZ interliggi_k_Page_081.jpg
88a30dc5484300980a9bfd14107c0101
65528b8fa5d5b9046720769e781b64cadeb3696a
F20101118_AABCEI interliggi_k_Page_019.tif
6c5f2a932e88588dbba5a7e866909a78
982ec5330021326410de00b951703441dc94a2c7
21426 F20101118_AABBZC interliggi_k_Page_005.QC.jpg
f37a9cf9ff3c33a629a6ad483b111517
9bb4a2d3a4cc3b1be66a7d84e54b0052f6eca967
28929 F20101118_AABCDT interliggi_k_Page_098.QC.jpg
8a41c236c9b59322ab21b73832d9126f
c1a1b32870183e921592fed641dd7c6017b2c2c6
39410 F20101118_AABBYN interliggi_k_Page_125.jpg
dea1759dd661a3b37285570b6f1590c0
f5b6a62101097b4d5bc6c297577df3f10830b1c7
29488 F20101118_AABCEJ interliggi_k_Page_148.QC.jpg
56909054aa08f02374389f355aa17064
12f583cf4f6907b2938d88c1844c4a5cc770692c
2672 F20101118_AABBZD interliggi_k_Page_084thm.jpg
be93235ff274201bb2109d749897a166
01192be09e8bb0bfae60fd82ae120ef7a1476fba
540191 F20101118_AABCDU interliggi_k_Page_077.jp2
bd841f907c2a6334e0a30095ff6e61e2
95174198321e429edc482391b2135c52086447cb
22419 F20101118_AABBYO interliggi_k_Page_140.QC.jpg
9c636895ac2b856448c635ba6e385836
64b4f1c87df642ebee4ba80c4af883124764de0d
14429 F20101118_AABCEK interliggi_k_Page_047.QC.jpg
ff3b6fc638fc7ad7845bb4f856e0580d
bcc8e0fea50debfcae3682beb49e3eccac29f49d
F20101118_AABCDV interliggi_k_Page_058.tif
16c2c8ab49ed7d8babab526db90cef7c
a3224565b69858e3cf707276c17e60f5bdc9d70b
269536 F20101118_AABBYP interliggi_k_Page_001.jp2
9e61d485d32319b3bdda5b19bbdaa3a7
6f27e7180010fa224a3f293dadb7219d1b736d8d
1051986 F20101118_AABCEL interliggi_k_Page_024.jp2
3cb44f2c29ad7b4eb5473e4390f96151
a92c649310e506be1dd516d2141bc0414ddc030f
1051968 F20101118_AABBZE interliggi_k_Page_136.jp2
89a1d0723a39bd78626d4258867562e0
ca43648ea56eb1a1625a6b3d6dcfcdb29dee8bba
4750 F20101118_AABCDW interliggi_k_Page_064thm.jpg
ee7fb7eb00a83bd7f5ce4623dd91bf2e
77333f108b2d8a7e6a801fbc3a97b37e7843f7e3
6171 F20101118_AABBYQ interliggi_k_Page_065thm.jpg
803a38a458fdd8731f74c08ef0a71513
8736209c1b8dd460667087957ef0d2127c3c68d8
377408 F20101118_AABCEM interliggi_k_Page_062.jp2
05a057f98bc2b73bb6c2483e29715a6b
c13779a7a6226e52d2033198970daab2dd931655
1051948 F20101118_AABBZF interliggi_k_Page_095.jp2
3832a79aeb7ff28084fc9fcbd5c2b0a6
de2e6cf357ea3d0431f985d7a2b0e8cd5102ce49
896936 F20101118_AABCDX interliggi_k_Page_082.jp2
ed5208c309d84bdf7e8ab5a22d651dec
7ce9ab4e53d2bcdf6b64c2baecabb861ae80a328
904 F20101118_AABBYR interliggi_k_Page_011.txt
f1bd0271798fc22874e1886771540393
8a7421091ef41d700d28a7dc16946ee7254ef5f8
1051950 F20101118_AABCFA interliggi_k_Page_014.jp2
18ec2c4d5789b2d67df69c0d7b6f6df0
9dd0b227c9ca6531cd06035b63cf1592ca033e49
F20101118_AABCEN interliggi_k_Page_075.tif
88aea3fd0ff6ef9b42d5041058288fe1
5c2613edc87b0307218c98268da93aff1d714676
F20101118_AABBZG interliggi_k_Page_077.tif
b50ceaf7f2f3b3d904f27b1edb48d034
577b46dbb89c6a354cc61b6973813b2d7b2367e5
18689 F20101118_AABCDY interliggi_k_Page_100.pro
12ed3898eba20170043e1bfe89e33e4a
748c7dfa05c6f4b5e18d6dd4b5b83ca6d889f29e
927750 F20101118_AABBYS interliggi_k_Page_127.jp2
239df8cd740f1e0fb851b6c3cb2cc4b6
7c192b8aa96629445184295e2611269c0ee61c69
42571 F20101118_AABCFB interliggi_k_Page_132.jpg
ecf4a4f73d075bc82ffbb9f968b0bfc3
7ef058aca542f80bb4059a400a826f8ce8c683d7
6096 F20101118_AABBZH interliggi_k_Page_025thm.jpg
489f984003cdc4fb8d840b044b3f888b
dabf14397623532b4cec8335fe0cf8ce79ca93d3
493407 F20101118_AABCDZ interliggi_k_Page_106.jp2
b16da6f1c78ab376e7d79c7d853fa693
a4e88ed3c3faf171522d5a278cbb30990da3823c
2180 F20101118_AABBYT interliggi_k_Page_135.txt
9eef49bbaf2b603789de59b286aa5fdc
7d55d32a2b77407d755b3a60a6cd4b13a6ac0768
867331 F20101118_AABCFC interliggi_k_Page_052.jp2
c09a54059e4cc0f6e5fdfeb74bb24f67
1894f2162aec79b143976d43978af6395552e771
381723 F20101118_AABCEO interliggi_k_Page_054.jp2
225002f07ed559422b7e277e58f5fc4f
7eab57bf1eaf338900ec8cebf4ad43d5e7f3853b
F20101118_AABBZI interliggi_k_Page_013.tif
00cd5ec7fb0d99dffbcdad050c5e5367
42a2fc8145cae3772fe5707f2b59b54fc6ecedae
8742 F20101118_AABBYU interliggi_k_Page_111.QC.jpg
2e6487f9ec47cc950c890b7bc6d8019f
faedc3df60d1e82946032376f15497a6e90914f4
49345 F20101118_AABCFD interliggi_k_Page_004.pro
8b6b43806a919df9540c9279d99f9727
3640bdb19d713cf87bcc3a8f089b2b76bf59a535
23919 F20101118_AABCEP interliggi_k_Page_040.QC.jpg
95d0ec8b3ebe791a02e36357225ec96e
a0d825a56d86b78b4e7168f608e018ede72fd8f4
27802 F20101118_AABBZJ interliggi_k_Page_071.QC.jpg
316f8c4cf7de6a11dd32f6f216b9ab30
a3e285ec7da760bdb719288eee1db24665499c0d
F20101118_AABBYV interliggi_k_Page_054.tif
95550d221b80d2ee562ac394030dc5a1
ca75a52d9555a90dd2f257e8b9b91cf7081ea898
383243 F20101118_AABCFE interliggi_k_Page_084.jp2
0002f68c787d8ef46173be315766b319
15f27d932254ac3adf92192f1d0ef636c4b34891
46916 F20101118_AABCEQ interliggi_k_Page_086.pro
58a009b1369ee0deac52c683b612a3e4
ef5850b79b8946c28a2178ec30bf4362af0fdc1b
6446 F20101118_AABBZK interliggi_k_Page_015thm.jpg
eb3046521f085911cc1abb3ac80d4797
56484fd36bfcb10b33ca54d8659faad4b104d397
2157 F20101118_AABBYW interliggi_k_Page_133.txt
dfc4c93f00f7b7cb950c4dd2a9a0a60c
1dcc86de346258e59cd07af44ac6c90eee0846e8
6358 F20101118_AABCFF interliggi_k_Page_117thm.jpg
a99539d8795162377b112d3bbd44ea31
3a797c35133a365bfc80db818ff1d68143b8327b
2094 F20101118_AABCER interliggi_k_Page_117.txt
80e90ce7db46220ac7824a1c5d4e2660
b972fc4d5a32c33751f39e865016a0ba3d85e51b
36745 F20101118_AABBZL interliggi_k_Page_101.pro
69c11b1c586d841df5f76c373081373a
5914cfbcdc9995a5873dacf9b853daec9b237cc9
6517 F20101118_AABBYX interliggi_k_Page_043thm.jpg
1989516933d70d5fbb97cf36511c5758
6f557a6b7ea69b0d9c430de0bccb7c1b0c83bd25
10973 F20101118_AABCFG interliggi_k_Page_048.pro
fba865dfce7b5f99cf4fa1e306066335
c1c34bc5f2790be081e9c08ca540e32c579876e3
52399 F20101118_AABCES interliggi_k_Page_036.pro
210a94dbbeda474d88288a90a34939e6
402a869598de57b9d46c4040379ff51ee04b0f6f
F20101118_AABBZM interliggi_k_Page_024.tif
aa00f5cebf8e98bfaa7d657cc2c153df
ce7f7d5b6e3abc750b676a96a36cc108411d8efb
61190 F20101118_AABBYY interliggi_k_Page_074.jpg
7ce2973fb515202c30e3626acb8cf05a
abb634bffcbd44e92991d2d531453e6475b29bea
820025 F20101118_AABCFH interliggi_k_Page_057.jp2
282d72b6872b8dd3351d74499d8e76ef
e476735e6b075bfc026d354e4f23ae91ff36a68b
1051984 F20101118_AABCET interliggi_k_Page_035.jp2
de4dfa56e22f8435c1efd73f6e3d57a3
c5828ca08a739ae77be6d207a5168ef38bf54c8b
626729 F20101118_AABBZN interliggi_k_Page_108.jp2
5e25c642819ff0baf0496b046d76b85a
174eb2128c85fc33e1ab0c62deadec08296cc5b4
F20101118_AABBYZ interliggi_k_Page_068.tif
3fd30368e3a5602099d6494aba72ff87
6000058367d8f6e861b67a2e996d70f1aa9bed0c
6536 F20101118_AABCFI interliggi_k_Page_070thm.jpg
ca465ed39f8614fb6ed0e4145f8defc9
43696c93318c4cb6460e530d00855a3fd8649b8e
1051974 F20101118_AABCEU interliggi_k_Page_073.jp2
25e6904d2798a53bd608610fefa0b72a
3b9c93ab151967f40e12e3f267690fa9f6042f02
99650 F20101118_AABBZO interliggi_k_Page_005.pro
2eb76df6f491d9ffffbf8947315c94f2
f2a799d3160c26d9f11e7e1e2171533b67ed60da
2022 F20101118_AABCFJ interliggi_k_Page_140.txt
89bbbd651f929e19226fe89ddd6e1ed5
56fba09fc9109e71f53fd38f2c201210d8322124
2065 F20101118_AABCEV interliggi_k_Page_118.txt
d6da3dd4e55380b06f6db13ad4a78677
fb74f01c40f6308c124d505ea3168ed201347abd
10309 F20101118_AABBZP interliggi_k_Page_132.pro
7ca119292105f04463fb784b513d39c6
88b7308d8ac048f2a7fe1fa29a70014014da0005
34830 F20101118_AABCFK interliggi_k_Page_064.pro
3381f998808a02304c583537e1fe03e6
ed9aff63b6ab8f971b94aa1a940e08a44ee443b6
6992 F20101118_AABCEW interliggi_k_Page_009thm.jpg
9db4d15f859c12973fee6d087a3da4b5
2f2cc20870a76452f3d9d792502aeb6f67fb23f3
F20101118_AABBZQ interliggi_k_Page_121.tif
677e4a8e06dd99c8b2b749cbaa5d282b
ee45006bfb2740687d19edf8412a3810265ddd3d
15678 F20101118_AABCFL interliggi_k_Page_010.QC.jpg
793bb418fb59d49283aebfe5eb6caded
3ebe1982f7bdfee7e5b0a01837b1baa3d432d868
939 F20101118_AABCEX interliggi_k_Page_049.txt
752b4406eb3d05f94b0b93633c7734a0
92bbc61a70697bb354ef93538a1a3bb6a1a2bfc4
1051979 F20101118_AABBZR interliggi_k_Page_016.jp2
a99d5151e141c282656c1d1dae0273db
9539df1afe45481ec9ac36bd1a16f15cf18c4210
5211 F20101118_AABCGA interliggi_k_Page_079thm.jpg
010e96a54df58f4455d931f487177e33
3d37856a7749e1ec08f9c7375f82bce2d0dd0128
1051935 F20101118_AABCFM interliggi_k_Page_071.jp2
cdddfee629ad9e74a7f8eb2ac911fdb8
2c07f258892b908c40ccf136771c5637bf529c74
26054 F20101118_AABCEY interliggi_k_Page_065.QC.jpg
526e6f5e13c662a912a176b3fb668fe1
d169da55de856babd3dc2ebeeea58a1d9ac9d2b7
F20101118_AABBZS interliggi_k_Page_121.jp2
6425086a6b957d094d2b4477e08d0b14
ff3a0a6608a325093510d79e423708f7b588387b
5269 F20101118_AABCGB interliggi_k_Page_005thm.jpg
27c693f4b9431e180b396be7de51ef5d
408d9d708084f5e4928e548a3d12fa331e9ebb9d
6107 F20101118_AABCFN interliggi_k_Page_153thm.jpg
b201c8249a72503c69d435b0e2ebeefe
1bd6d16ee918f305855fe30086efbedad5725c0a
1051845 F20101118_AABBZT interliggi_k_Page_049.jp2
9af1bcadf0acbe851943a48e4e65ecb7
90eff6817f2102368b27815c6cd3c6cd677c748c
1051976 F20101118_AABCGC interliggi_k_Page_093.jp2
bee4a4efc10c31d8e4e20429c501fc69
d2e0cff0a79cacd32108077c6dc592323dcc2ab3
F20101118_AABCFO interliggi_k_Page_131.tif
1e69f1861eb0f444fea27b84ddf27077
7b5e1cc18e456f08ad266ddddabdd8ec38fe9b0b
F20101118_AABCEZ interliggi_k_Page_088.tif
98fb6d54939f235712aab47763f8da63
e24435008dd358bb18896a50ff5c283a7eb1cf3a
91576 F20101118_AABBZU interliggi_k_Page_038.jpg
f6fc0d62ba52e561998b6af0f0f57e39
d1c6fa9e7641076b90ae1e64557fc2001698c99c
35280 F20101118_AABCGD interliggi_k_Page_103.jpg
bcb0b86999cd050b15b167712e04310b
01d797cdf7e6ef95c6ce4902187ae659621d8a73
1051978 F20101118_AABBZV interliggi_k_Page_059.jp2
52f80ccbd950a1f480bf3f4095c79bd1
54475babee0c1aaf6da5b83a9375a54667e86e6f
717125 F20101118_AABCGE interliggi_k_Page_126.jp2
855bc0e844312ec67430e63da1a441ff
a34eca498e9111951d30c33343978e74d7a3d12c
28502 F20101118_AABCFP interliggi_k_Page_106.jpg
aae2fdf0bc3af4bcbf589c2a85acfeae
6dc2deb835152c7a9dbf4d6ad3792b39334d4cef
6541 F20101118_AABBZW interliggi_k_Page_120thm.jpg
a8a94399b84b65b861d756fb69006f99
dcfb7498dbb10bbb3bbcd979f8d0f53ab1bf5632
5774 F20101118_AABCGF interliggi_k_Page_012thm.jpg
760940dcd5d2194b3e6ea5b4faf6bdbb
a7c4a457ad916f18f066ae4e705a67ed314b908d
88214 F20101118_AABCFQ interliggi_k_Page_015.jpg
46737462378b273bec0a83daa95d5bb0
153b270a686a6b71a665c377befe8da377e9a5dd
1067 F20101118_AABBZX interliggi_k_Page_047.txt
dec788b9fd14b0d52ead09acd17ce7c5
eb61df7f05350c812650040c6460b46341be4dff
6406 F20101118_AABCGG interliggi_k_Page_045thm.jpg
214de3725a61dc4a96e53a865aa2121a
6c99300654501df8b24c252d05d2a0c93fcba0e5
6753 F20101118_AABCFR interliggi_k_Page_097thm.jpg
435319cca9246603ae12a611c7cbcc41
cc0e93f72fbfe580073d34f183baac8a63d9ff23
43972 F20101118_AABBZY interliggi_k_Page_100.jpg
4d3907af3f06e1ce4939c8768e2dd724
5f47c4ca0477e52cab0480143eae030585346b60
2225 F20101118_AABCGH interliggi_k_Page_097.txt
d59aa24e8f592740af354d7c77b66fa0
7a5a234f8b21fa8f1db3a1b1e11f4a867cf8ff5c
8649 F20101118_AABCFS interliggi_k_Page_084.QC.jpg
777c134420ba33526b9e8a9d78401909
029fbd12b8e7746a148c85cbb96a7bdbdf53980a
921641 F20101118_AABBZZ interliggi_k_Page_055.jp2
71f0b04c7d4db88de0ae4eca2edef3d0
d2672e545808077f57b371e534edb95001a33e47
F20101118_AABCGI interliggi_k_Page_115.tif
df9e4df4ec53468638b55b6d2ac44bcc
60f0a2eee36582a2fb8fa4e2f350a7208270fe14
6492 F20101118_AABCFT interliggi_k_Page_004thm.jpg
163e0fc91e0663278054da1b7d5554c2
a1bb2c27f998ce3e350d3817eca6963c3a42c8b0
52220 F20101118_AABCGJ interliggi_k_Page_133.pro
ca65e3534c6f20f55d72a0a7e72956ec
4c0abe0a87d9e65892177326e1b1cbb750bdadde
89455 F20101118_AABCFU interliggi_k_Page_094.jpg
3939ee17edf0fce1cd8c64d03b818a9f
1d1e3724c2effbb23373a749a3e8978228bf9426
F20101118_AABCGK interliggi_k_Page_105.tif
8a0e881efbf7128722ca664889cc3dea
6e85c285df7e90e6b38617686c13b069c7f85fd1
34856 F20101118_AABCFV interliggi_k_Page_011.jpg
d661367ac8122136c383ef960b26388e
b1ba765e373103e98874eca4def920e18a850ed8
6823 F20101118_AABCGL interliggi_k_Page_044thm.jpg
977b1e5e78f860343bc5dd3cecee5999
00df17044329e8d6edbd6d1a2afd01a4e1d5f616
F20101118_AABCFW interliggi_k_Page_042.tif
76dcc52dce6c73ac3b94d56443f0d1e1
679fde36323328f81d234890e6e9e2814d87e4b0
13096 F20101118_AABBDK interliggi_k_Page_031.pro
62e908ed46c4240d5ff559bf704ceba4
3816f747e2c7c255e486afc3ab942455da86aba5
26431 F20101118_AABCGM interliggi_k_Page_039.QC.jpg
d050f82d41b317f664a4a3bac5f05821
b784bda7f39b19cf69a8f918be88102bbbbce66d
5862 F20101118_AABCFX interliggi_k_Page_023thm.jpg
2aa5f60e51eed09957a9ba0754fa8bfe
58aa3dc4c9621cf64300635f1fedcdc913bce23c
F20101118_AABCHA interliggi_k_Page_053.tif
2097543a1158714200ab56329c3d3e1b
38afb3e4ffc6466a68e7c22c5a610e4439ee00a9
3654 F20101118_AABBDL interliggi_k_Page_125thm.jpg
066811239d26f9273faf9870c6ccfb37
a7f08a883f119c996b6a6a6a4c639c9d3b1a0e52
474 F20101118_AABCGN interliggi_k_Page_002thm.jpg
e09f2b151e2808a55999dd9cbd95cac4
b997e246353412788d9e1e3d78d63c80e5adc6f3
1284 F20101118_AABCFY interliggi_k_Page_030.txt
f7427eeef8a2d70a186e002cd613f1f9
b574b3076ba43c701639b7b7055dbc9569d175e6
89763 F20101118_AABCHB interliggi_k_Page_072.jpg
08acf0e59e0b4f00661f8526064cbe63
ea9e66404e60e42e4c5c0bea78f18e1e3ee9b75a
8128 F20101118_AABBDM interliggi_k_Page_154.QC.jpg
8d324a6bd09a62d525a893ff78602f88
f5f1d0e321aeb4ecc1bc23a940677e6e9d9e4216
2990 F20101118_AABCGO interliggi_k_Page_058thm.jpg
af7af903a98b59a00f696f7a45079642
76a0d3a85a68a92a2d98f454cf3cc8e8524a6d56
7490 F20101118_AABCFZ interliggi_k_Page_061.QC.jpg
a7cbb0c372eed64bff94131774464948
11500f3f25bd8559f13de36373a8cf903c1226f0
1023297 F20101118_AABBEA interliggi_k_Page_063.jp2
ce07719903aba9947b4ab4c662361d80
083d7d91f0bd71d50f5aac5739a8716c5da2d769
1051962 F20101118_AABCHC interliggi_k_Page_115.jp2
a43a889b7d4579903fec17c0af519daf
6b99bd7a2536bfdb252527d82dbeddde8d381660
F20101118_AABBDN interliggi_k_Page_133.tif
a6359b1750cb4487299241762b8994da
c233453f606e49d277060cfd7aefd55e1fb4d0b9
747982 F20101118_AABCGP interliggi_k_Page_107.jp2
fa507d80f4bfe070c882b86cf96a5bc8
28bc5970c8394c819a7be9b72575c26783dd13c7
343365 F20101118_AABBEB interliggi_k_Page_111.jp2
99ab891a050bbf8026ccacf08b5c4875
f64942a8bb4a3944e70186c6cdfe0337c058baba
4438 F20101118_AABCHD interliggi_k_Page_006.txt
c6b1a3051e13bcea2130bf058eb35f1f
b47e0c2a515cc80b4bf91b0b130d690e133339a8
F20101118_AABBEC interliggi_k_Page_006.tif
3af00e816b940d11c9ebc3aeac1f1796
9a41579b8a374a0aede255c5b7d11ad3a5eda2d7
4083 F20101118_AABCHE interliggi_k_Page_046thm.jpg
0cf0cb55ca837d8955db0cf7bb5aac06
6bb9ad830ab2cf770d3a8c43486031169f7cff6b
26965 F20101118_AABBDO interliggi_k_Page_113.QC.jpg
1120362af9f77369c1a3dc024966bca3
5b8bd5d67fc8f4fd593e9050eaa7f1d939c835d7
29262 F20101118_AABCGQ interliggi_k_Page_030.pro
1223013929cc42e33924857fa208291b
7bee7b82c0b85ada26910f1dfc77fead92e4ff14
2025 F20101118_AABBED interliggi_k_Page_069.txt
c6d5d9981787d4c956a4a64da6ac82c1
729e0fce2a79324497097afb38a1f88ae7d8294c
F20101118_AABCHF interliggi_k_Page_095.tif
edf212add7f4f7931093012b9c8e82e4
e0318545b17c976d026fbb67c8c48f690a123952
1051970 F20101118_AABBDP interliggi_k_Page_033.jp2
8695b577b1c0bca2fc7d2c5b5d2c05c7
92fec21e36f17d40c285c81e793286677a0cdccd
F20101118_AABCGR interliggi_k_Page_059.tif
66dfd1a9cc12a3c8cfaa33ee0c0779b4
ebd8cb2feca26ca8cb7d7508bb43b6f029b8dc07
73328 F20101118_AABBEE interliggi_k_Page_020.jpg
c34f810bde3e501b7c1428357f6233d7
34b2c4f2b36ecc362995af23d5abb8a844de50ab
521 F20101118_AABCHG interliggi_k_Page_110.txt
c657f89d8a37bc402fc2dbc5ce332f25
8981d0a52caca9eecbdea87f66c8a0bb7ea8a761
F20101118_AABBDQ interliggi_k_Page_122.tif
dce721bf29e44ec1fa5cb1fa2035c93f
2047a747b88e2b9594e3aa2b7e3cd528e6db91e6
F20101118_AABCGS interliggi_k_Page_130.tif
946f0bf6a25cd940135152ab44398986
05744cf3a08184100c8e3aafc29a58992ccbab8d
89579 F20101118_AABBEF interliggi_k_Page_091.jpg
aa856ef3c16ae61150336a806e5ffaf0
efb0a5bb2936c1dcdcda6ef81735e8a7ada12f7a
10069 F20101118_AABCHH interliggi_k_Page_081.QC.jpg
eb632fe1dbb3d021400e733a11eabbc7
6038dbcabd08011bcdd5ea2e7d78855c38e0dd62
248 F20101118_AABBDR interliggi_k_Page_129.txt
7fc0647dd56516e7d55bd55fd59432b6
a4269458d968a0c50436d9158046f5a6ef978ef8
6676 F20101118_AABCGT interliggi_k_Page_090thm.jpg
17b5d229574b48312d50a629138f0d8a
c565afedc8b997cb6148b2a1bb4a219e7a286042
55829 F20101118_AABBEG interliggi_k_Page_092.pro
7dc4e4b5a380439801cbeaf04d5f4263
4b1c068227243b7b013383d2eec007b3ab8086ae
51870 F20101118_AABCHI interliggi_k_Page_119.pro
4f65d58c7f1938fdea6d9db9682a68dd
66dd604426b1218e30f745c8f405c2d767405e75
F20101118_AABBDS interliggi_k_Page_032.jp2
0cc81b14fb8b128db609ac4f3662b5d2
b79ebb41c8a54639c6471441d8293e296d1cb6f2
55574 F20101118_AABCGU interliggi_k_Page_137.pro
68aec11578509f0243eb6db424f5951c
71e2049f68e21f2fe62ae7305e4c09b186268bb4
2114 F20101118_AABBEH interliggi_k_Page_120.txt
cc7ac0bf5e13598684dcaa4d2fbc5a9f
9663a7b0aa1db16b8e5ae09640131c19b709b779
54142 F20101118_AABCHJ interliggi_k_Page_083.jpg
66099b985f52372c52680c7d0fe65f3a
14c12c984ac12169311c390cf7b7dc0e1147484b
F20101118_AABBDT interliggi_k_Page_009.jp2
b7b7b0bbd2ac3844bd644466c638ba64
e953dd6ccef08b36a6d5ea0d5694617a677f7582
2179 F20101118_AABCGV interliggi_k_Page_072.txt
3c612fedc3d15fa2f52485f6a6c40895
e1a8143b6a5d4144ae95783fe2a05f12811d6e02
25910 F20101118_AABBEI interliggi_k_Page_028.QC.jpg
dd180a67c8261d904ddeed0f0488fa22
5c4ca6a06fc4ca4b0331bb35c8b44d8eea01377c
909468 F20101118_AABCHK interliggi_k_Page_131.jp2
56ab4a5e31d9f60ef6c6ee198d6c99ed
8b5cbd0cf80e12e8e4acf12bba71dbf957cf20ef
F20101118_AABBDU interliggi_k_Page_118.tif
b88dfa083935accd3bbcae86c39b1240
7bd704d2694b7e8e727907b1b1a6420ee6ff7fd6
6205 F20101118_AABCGW interliggi_k_Page_042thm.jpg
f35c3bac08be5eac1bd373549beabf59
07a9180c20ec944ff85d5985fb5da028908479a5
F20101118_AABBEJ interliggi_k_Page_126.tif
d742766c30a1c0e26f7d31da840c2c06
1f503a8dfe559b0c22e8e8df70b2df93e2c8ff29
80704 F20101118_AABCHL interliggi_k_Page_139.jp2
54f0c7e9a0e33fb5514323e45fca6252
5205a88049de2d2ed43d305c692880dd2955117d
F20101118_AABCIA interliggi_k_Page_041.tif
c3557645f023037c26c7ec973eb77096
0b04c0716fa4529197f97eb5f3273780f3b09c28
F20101118_AABBDV interliggi_k_Page_030.jp2
2fbb32eefb2bfef7cb474abebb8f16ff
a6d1b4071ce086882a7ddd8cf8651b89677835f0
96901 F20101118_AABCGX interliggi_k_Page_005.jpg
8e389a352199722fdc0ed0f7bc0dc758
6acba4eb5d6cc6f0d3aa9af11167bfaafd48c43a
F20101118_AABBEK interliggi_k_Page_011.tif
ae242dacf4fbd0c23a305e4d06726144
d80376789df899f6bce0d6af4fd2cd6beb9c53ab
60955 F20101118_AABCHM interliggi_k_Page_145.pro
d94f6b78c29ef215193e41e4ba03719d
66310d5ef73d21ceb937e277965e997039c7718b
24461 F20101118_AABCIB interliggi_k_Page_059.pro
fbb3bbbd3c9645c43fcb7ca98bcc9cd5
1af03ec95030530a9ea8f2b84c7de2d69a783aa7
F20101118_AABBDW interliggi_k_Page_010.tif
055dc270dde91f19753c1597882342a1
cd5f8512d005992ee90427c8d712a46a65abceef
15238 F20101118_AABCGY interliggi_k_Page_127.QC.jpg
ed0ed8ec091534f96a1f3bc9e8ca69e0
6956e8f8b7bb3eb281385b89f195bfedc317f91c
F20101118_AABBEL interliggi_k_Page_087.tif
573fbee885a33150d61a1fcd8f730799
66f27ef0f5e87803703746a29f4755ebb7238ee2
F20101118_AABCHN interliggi_k_Page_030.tif
d2ec108dea896f16742f2d8b4af4d015
182f35fd13ac794a93d438260e3e254410c70f0d
F20101118_AABCIC interliggi_k_Page_071.tif
4b626eeccaa105bc2929db859e9fcdfa
ee0996488775f037aeaa188d4b97ec8bff542075
F20101118_AABBDX interliggi_k_Page_029.tif
bd7983526c14a6f6c02eb936ca000e07
55e3bcf817d5f1fd8d3cd16f68bb5421c6032b5e
3738 F20101118_AABCGZ interliggi_k_Page_056thm.jpg
1c373122ad29518e3d657e26415e982b
f3f39afb1340d7d8faf5d23d7f851e4716976e71
1051 F20101118_AABBFA interliggi_k_Page_059.txt
85da724775429bb4cee541a393af6208
9789ac2840411147524c29c0070f20a4a676dfec
6950 F20101118_AABBEM interliggi_k_Page_072thm.jpg
98516d0524179efb6ba9a28f2494a07b
f23084cd6dfe13876bec9dab2af17c10f7788bde
F20101118_AABCHO interliggi_k_Page_135.tif
11b43c0e7d2ba8045cf55eea5d3307fe
b905e5f22d69bebfe0ae5d741b6edea94a473a9c
1051952 F20101118_AABCID interliggi_k_Page_018.jp2
b0b678b4777b843e3a81eaa0946fec9c
f432cb6ad1a0f6291ef9e472126efbb9339e9cfa
107932 F20101118_AABBDY interliggi_k_Page_147.jpg
e332860b9b0715cf0f50847648d947f1
53e91165498743fcff81b1ee4579c7bbc38b4c18
26690 F20101118_AABBFB interliggi_k_Page_119.QC.jpg
4f563fa747f1477463aa3dec076a04f3
4d37e4594e9bad63ad4fee414be31eb6ad9bd690
27682 F20101118_AABBEN interliggi_k_Page_121.QC.jpg
ea2160784096089f62f049a07038f83d
83569fc5fe27244e17022b83672d0a9e6bb1c6cc
F20101118_AABCHP interliggi_k_Page_008.tif
d4e8af9f5662865c7c35ac5cfef34c02
383c7a7636568b29f69bfd4e21fb003ebf7f8612
6554 F20101118_AABCIE interliggi_k_Page_095thm.jpg
e9c345ce5a5fa53466d59dbe52a02f3b
86f0151617092ed44c4b0d84d808b0b0cc10a370
11297 F20101118_AABCHQ interliggi_k_Page_125.pro
1334ace18914d25a87373ce2820b9384
d78d055d61625aa6783901fad6a3827073882be5
1051983 F20101118_AABBDZ interliggi_k_Page_043.jp2
81392841a63f4fabe3bbb560ec74a454
955eb6bf3f14519e58bac660a2d5dc19f30cb817
58594 F20101118_AABBFC interliggi_k_Page_079.jpg
a6aa51bda88fd2f4ca98371b78279797
c653b70b5afad22415aaa04f035863f9816fbbb9
10257 F20101118_AABBEO interliggi_k_Page_123.pro
8f13ba3b36f6e37d7036e0d17191e964
8a8fdc864412e3cad9e7e175c464f595dd8d6550
9864 F20101118_AABCIF interliggi_k_Page_031.QC.jpg
e2857710a8bd231f9fe2e96fb2b35a90
8084c0776f2ee91fd660597a41775fe52b653e04
F20101118_AABBFD interliggi_k_Page_135.jp2
e6479d2dffbdcdb5158ea8b1e77955b9
dd3987b15ddec67317f7041a109eb6d775dcf1a4
3270 F20101118_AABCIG interliggi_k_Page_139.pro
03138e176e547773637090e82ceb7958
ddb6ea2ba5b439f98cb5388a33d8fa64d343b25e
2000 F20101118_AABCHR interliggi_k_Page_028.txt
fb295526ab9e842099758a0e22d84595
c9f67e65e6f90b015bbefb0d5dad2c6465f93178
9763 F20101118_AABBFE interliggi_k_Page_060.pro
16b387c20d77802ea9bc1dc6c02f6d32
d66d0cdc2a07c00f1561de94a466bd4311e619ce
F20101118_AABBEP interliggi_k_Page_068.txt
9b93d9cdcd3154aa7eb28700fe647c67
4d94d6ca3773301a03dc3867f6a409d8eb4aa6c3
28462 F20101118_AABCIH interliggi_k_Page_134.QC.jpg
01ce07749bd4019eedfb03c2a95b5b8a
96657803e473783f028314ae07f0adf3f86ba014
58126 F20101118_AABCHS interliggi_k_Page_153.pro
47433e9944d13101c82ded681ce8d2c1
3b834930f784be36f40ebd286aefab11802e7550
71416 F20101118_AABBFF interliggi_k_Page_009.pro
31ac2d803b4ef61094374fe3c9249e31
c1ee44966ad79ecb053adab7d32acef0212a3e79
2160 F20101118_AABBEQ interliggi_k_Page_094.txt
64417bb097214673e00139cb7ba6722b
51a1637e89c20bcdce48106bc0da149b64b4eb6a
6682 F20101118_AABCII interliggi_k_Page_035thm.jpg
b30d7a32e4997d6f8ba0f409d58ec271
b277cd53e45321e05b18e1c9c04fc383b6ece2a3
815224 F20101118_AABCHT interliggi_k_Page_074.jp2
22426f79aae5822f6ed79dd83ae6c512
d6eb634ba5b8fabbfb4c81aff95a37938eb5a7f5
F20101118_AABBFG interliggi_k_Page_050.tif
7a1c9436147a4fae3519b0fa8f70d61b
1a1ec9dde5b5e02678279c5badd080115c8970f8
3010 F20101118_AABBER interliggi_k_Page_102thm.jpg
dcc67eb7ae690e1ad19eb8337a5d4430
b612eeb1676d36761cf214bc4413a942697e2cbb
F20101118_AABCIJ interliggi_k_Page_015.tif
664ba3ee5d2889b0b54dac41d29f6f62
9ac4d3d1fc25a7dbc852d0318b7be3d5931a22a5
84148 F20101118_AABCHU interliggi_k_Page_039.jpg
6dc181b4328d32e624f049f72cc5305e
9ee8f5175dbf696834bdb8f5fcb0a4ee74155e68
2142 F20101118_AABBFH interliggi_k_Page_071.txt
693328be51ef2c9c7a045919068818aa
fdc850bcdd9acdaca5370625fd0147acee14775d
1926 F20101118_AABBES interliggi_k_Page_053thm.jpg
21256cff3f42d27def6834cf0ab24168
28f987fb3a90e922218efa13b6668c393233d9fd
2773 F20101118_AABCIK interliggi_k_Page_003.QC.jpg
357a27501f98db98bcd1399b5b0c6606
b91182c781261a615e0cf07271fe9dbc96ebfcd7
1680 F20101118_AABCHV interliggi_k_Page_099.txt
b7310c2da7484aa3c31d727cc5465b25
668617f786541f3089775e9a2ca8b4bc080987cc
493367 F20101118_AABBFI interliggi_k_Page_078.jp2
c3ac5118e0d7d5db07a7008292001f89
b119703bb4732e17e29915217eab2925b66049de
88594 F20101118_AABBET interliggi_k_Page_035.jpg
ad4e566f647407704146a2a4bd45e1a1
c1a6f5f78403128f3cbc0349e6647db0dfc37082
24463 F20101118_AABCIL interliggi_k_Page_130.jpg
07679acad9eac2d0e5ab0ec5202f47e1
45d24e8aa2b031bfbaf8d265189857953516f0fb
8812 F20101118_AABCHW interliggi_k_Page_054.QC.jpg
37c00969dc8b213708456a2c86001727
4bf414daeb7f6fd6f732251ad6901296c84c96d6
1051963 F20101118_AABBFJ interliggi_k_Page_150.jp2
7fb95b1ac9c63ae03e2792c0184c15d7
a5db15e5bb96b06ed256b704032e12972f700ac4
F20101118_AABBEU interliggi_k_Page_084.tif
188167160c9392eb0db92530cf361a6c
e52c7821cf1a61e0c98237a366a7754267d70c4a
681 F20101118_AABCJA interliggi_k_Page_112.txt
e1dd89d95b698c256995a15870568b28
60054c4ea477b83f8fd436c32975c14118211586
1051938 F20101118_AABCIM interliggi_k_Page_138.jp2
ff59e8bae081f37d4b4e9bab0a05eca2
56c3d2b79fd9ee6a187c333e1ef6e2207ae360bf
2244 F20101118_AABCHX interliggi_k_Page_138.txt
b32ebbd1065e9f95e6f07de775c52433
b26db00dc82f98ac22c98378f255c56c9c64adb4
769453 F20101118_AABBFK interliggi_k_Page_109.jp2
4030be95d7b54b1f100158263a316bfb
fd0f6127e005d186556820e202fca908b7de3f78
F20101118_AABBEV interliggi_k_Page_148.tif
8280d541a79b5fda0baf3e90918f560e
781afa885e885e500c63a747b2d5076fe6f4f2b7
5608 F20101118_AABCJB interliggi_k_Page_006thm.jpg
cd67ee246c68199817bb4a053b3a67fe
6b3007b66ce24735eaae2f8a74e1f5efa3310499
53103 F20101118_AABCIN interliggi_k_Page_021.pro
b1e96b41d3fc5c0f02416f0fb4ada4b7
ecfbf12ca3e2320d093f26a3dc4919e665c42bee
54682 F20101118_AABCHY interliggi_k_Page_046.jpg
18ef124e0aa782a7459b7b0252bf0c81
79f875698d3bb24c34e39f254a31e763ebf79ab3
985 F20101118_AABBFL interliggi_k_Page_083.txt
cd1bca84abb307231467257a6d81f905
58391b9c4f228244726e42bea914507ab63e95a8
F20101118_AABBEW interliggi_k_Page_111.tif
2922e48b04f331f7c45d0a4b23261e7d
df22f49b7d35bb75da072f8ee6b16022aacb851f
23345 F20101118_AABCJC interliggi_k_Page_085.pro
86979989db2103c294a2652dc287b963
3a15fc4db8a71292d9bc7f55898736545124b244
F20101118_AABCIO interliggi_k_Page_081.tif
9aa8671600658b3e0fedef7f71ba6d92
3dbcfaa94bdf84b8f1455877a4bc5e19b5989ad8
F20101118_AABCHZ interliggi_k_Page_065.tif
fc596e9ac680bba0acbef93d2a056e8d
8d190617e815cc647ee7f7fbc0fd2a92d5d8787b
492 F20101118_AABBGA interliggi_k_Page_111.txt
f16252ca4de4729f20cd24c5ff2ae34d
7b35999569a257962a0ae3feb9924c868c78d5cf
1051960 F20101118_AABBFM interliggi_k_Page_005.jp2
0d99c2d9288647bd9080e3d9cec67b6a
6e4f8a6783601af01bf273e0483b9ebf40b1454f
8797 F20101118_AABBEX interliggi_k_Page_001.pro
58ea45f89dc2bde604a543b96c5b94da
b48027fdc663f108535217ac4bfe35b2f512283c
27848 F20101118_AABCJD interliggi_k_Page_022.QC.jpg
f39160d55a514e5109cba52979bb7d44
62163950f9d283b2c68622013c75bceaae3ac06c
F20101118_AABCIP interliggi_k_Page_109.tif
8eb825994f860ebaa7dbad094f1c8f7b
0c2aad46632b8b94eb032a81ee8dae60724856bb
2030 F20101118_AABBGB interliggi_k_Page_067.txt
00040cd2369507c8c1f1dda20b6f2888
7e7f855f78362f59df46a2b979e6739821aaea4f
7169 F20101118_AABBFN interliggi_k_Page_130.QC.jpg
6c108e6d4b9760551db61825593f9a84
6f0bd3ebce76aaa80548090371170ee599c1e70e
1051982 F20101118_AABBEY interliggi_k_Page_147.jp2
8707009ece0d224168b98b84a8f0046b
69e3314bf644a6e05abdebfbe01d6e4fc3f277ca
90896 F20101118_AABCJE interliggi_k_Page_137.jpg
e0a2450d78265281c696b8a0d9f10e65
4d49d5c13f664de4fdf2f302ad06492f42f1355a
F20101118_AABCIQ interliggi_k_Page_062.tif
6efb032cd6a78f2649e49259e96c94bd
2d1a1ca0f7aae587548e5addbc7cebbc6af390ad
1579 F20101118_AABBGC interliggi_k_Page_127.txt
b0e482fd1a599fb43386a605e827d956
8e0ab213a7f0b1502c3286b2322d4f6f96bcc54c
6673 F20101118_AABBFO interliggi_k_Page_038thm.jpg
4906c3d1620948b6b3665b539dbed15c
41476402b7b6a409c3d957fb5992d1481dd7a033
107219 F20101118_AABBEZ interliggi_k_Page_151.jpg
8ecce848f9f9e0b16840391881595df4
7a09322cc9c044fe662b40d945b5bc6d2f8e9671
F20101118_AABCJF interliggi_k_Page_034.jp2
68d44ca45af735c9a85765bec38fa3fd
c6a19f5385cc3a555517fd84166bc00e3a9f3376
40592 F20101118_AABCIR interliggi_k_Page_060.jpg
2024815d7425abc79a54d73b1aa889d7
2b07cc06c9c1df81c0362fa8b8c94c31ad140f37
56387 F20101118_AABBGD interliggi_k_Page_056.jpg
115f437eaab705e9923bd23da418f965
830107ad7d98b7e4d3461fdd6bc1168df30965d2
6364 F20101118_AABBFP interliggi_k_Page_066thm.jpg
e1bc95b350904df58a14dc63edf92786
7b964a6920dedcaa046983d52ca4d98d4c319e7a
F20101118_AABCJG interliggi_k_Page_113.tif
53777dc532c4744f2e92e1581022deef
5cd180084766d11c09c74dec64642383665a5f6d
593861 F20101118_AABBGE interliggi_k_Page_100.jp2
e9fc4a01de94576da547fdf594eee79e
0621f48a60ec6dc1095c1a3f2223e9e9022dc26d
6686 F20101118_AABCJH interliggi_k_Page_091thm.jpg
19ecb931e6af365b85cd3dac5d09b78d
6b54a5467dffe62972a494a87a5170bd7577f416
589 F20101118_AABCIS interliggi_k_Page_102.txt
e900c2faf78d74c93580af8bafe186fc
b8e880da51b109f9770a5071491a68f953250259
F20101118_AABBGF interliggi_k_Page_092.tif
4d16731066ffb1d7e0e445b003cffb3c
c62a461bd71c05a76bf7ef73f9283b8fcda8be4e
F20101118_AABBFQ interliggi_k_Page_057.tif
d0404cc3fb81d012f236c0232eabc228
a2c7803edf4203baacaef510a34ff335ee3e860e
1056 F20101118_AABCJI interliggi_k_Page_122.txt
a92899c07825bb38a2c333221e268cd2
f31b5e35a6899f1fdef89074cf136ee64a520adb
6568 F20101118_AABCIT interliggi_k_Page_093thm.jpg
725f9adab4b506c524030c8a64569ffa
d8f1b47f028e78d12e5b0e30787f1e7d8905f7ec
86101 F20101118_AABBGG interliggi_k_Page_032.jpg
7665f8b03ae80b39335dfe09726a32e9
7fad206501023f309ab1e386882c45a65e125c0a
F20101118_AABBFR interliggi_k_Page_039.txt
bb1c8d85c2d6721e19f4232b36e7f8e5
2eeb9b120c4c3a647f308119a1b408e0914ede1c
77177 F20101118_AABCJJ interliggi_k_Page_063.jpg
5e585e609d4b09c4f77e10080c001e4c
6f0093089c43f92283b4ca1c9c4d973f83579c33
26514 F20101118_AABCIU interliggi_k_Page_014.QC.jpg
0b97fe30d1d7a242e4e6ca8773985747
4602c3075571991679d1c88d94c3879e3dde95d1
1051969 F20101118_AABBGH interliggi_k_Page_065.jp2
00ca63c34da1ad75ae938f25feaef509
0603e53e08df9697f609bd9d571e1b86de80738c
F20101118_AABBFS interliggi_k_Page_152.tif
d3f63c2c6560722e6f71d48f6a62978b
5297f3528d88e578fa6444bee45dcf0374e0b943
110869 F20101118_AABCJK interliggi_k_Page_152.jpg
a16b5fffe506a141bc6db08756fec835
b64e45229c0451b6b23e86a95ebc39abad0ddceb
104340 F20101118_AABCIV interliggi_k_Page_148.jpg
195671833fb2a1180810139f7fca8e33
fc1bc3c0624a07d3d4be3b2106a6cab452a00a83
1051980 F20101118_AABBGI interliggi_k_Page_094.jp2
9dba6768e7ab3aacb378677df33816c8
fb6c30601cd1a4a510c8f658c6b0472d61a104bd
2013 F20101118_AABBFT interliggi_k_Page_012.txt
22bbc300b624be97c0f1c85677121931
bb896bb7bce334d11e09d1b86a56cba4d32faf11
53775 F20101118_AABCJL interliggi_k_Page_121.pro
45f8a2ac6b552b380cc04758b3519755
7b16b31fd6ff26a9b368a7bb0420077450a582a7
F20101118_AABCIW interliggi_k_Page_017.jp2
0fedc93b17a87dad79b132971415a784
5a35d39115179215279be1e2c17e1ec9447810ec
F20101118_AABBGJ interliggi_k_Page_120.tif
385d1e19192024cdbf57600ffb62b593
ed28690e9038dee66834e9cb8ce865f07b6c5079
22256 F20101118_AABBFU interliggi_k_Page_107.pro
93b508b2bb94e5dcd4d20e6176a91e9b
b5e836d99e91d262d86282f498586d9f965079c9
22107 F20101118_AABCKA interliggi_k_Page_099.QC.jpg
13593adfb92da788285fa1c34977c918
a98e0e1000529db4ccfbae2349dd251a5033d4c5
5865 F20101118_AABCJM interliggi_k_Page_037thm.jpg
2c2e90944bc77430bdf7d1380a10edd7
b647b42ed5ce1c127bc7cc0348291ff065c3e65a
554004 F20101118_AABCIX interliggi_k_Page_125.jp2
eb3a038d3aa59209d0f773bd56c14f6b
79e5b515c28d71d9d1f4d329cafc70c0f2f3e8a2
F20101118_AABBGK interliggi_k_Page_064.tif
bc0720844e263ec7bc0ebb352c787a20
74fbf62832818396fc84d2b15f61d60e7f747003
51713 F20101118_AABBFV interliggi_k_Page_039.pro
09416641f670e221176824ee41e8abfe
35a57eceeef8e6788f777dfa4c6c909555882b94
1051985 F20101118_AABCKB interliggi_k_Page_050.jp2
1550e2624a917ba7aff2e90a5f1b9ae5
c4302ac349caf988b1ea6c3f50184c44c4ba75a3
36241 F20101118_AABCJN interliggi_k_Page_074.pro
32a0e210ecb0118b31806c6fef69bd71
e400b487daffe644d16e4e0fa301e7474e53b4e8
54948 F20101118_AABCIY interliggi_k_Page_094.pro
b587e27f018267d6fcb22d7dcfd9840c
5f6b2c12735d51d4d4409998a51555daa499fa84
34213 F20101118_AABBGL interliggi_k_Page_007.jpg
944cb1cd3c26f1e40c88ffe704598af1
beaf232be3f77d784d00270cdb00365fb6ac0d3b
433495 F20101118_AABBFW interliggi_k_Page_011.jp2
61daffe7b54b8bde0f166bd98fc6aabc
b27b56c4fefff03ba6cc1db6118be9cc9ec491bb
19230 F20101118_AABCKC interliggi_k_Page_074.QC.jpg
1da7edded411e6876f055074c2f3e476
3e7cf65a1e1cb32124079dc2b3e87c8601b2f49f
51113 F20101118_AABCJO interliggi_k_Page_069.pro
a93878b79cc4b9b4116cc2a53d677c4d
77eebe36d4aaca08144c30b0e4e363ec67f66684
51913 F20101118_AABCIZ interliggi_k_Page_041.pro
e809b1fdc5be162e55ada6d4e33351ce
e38a9be3739a85111527b90bbe9cd48432f79b25
91273 F20101118_AABBFX interliggi_k_Page_092.jpg
a80c444b7af4aa40ccddfd97d54c345e
f2afbd42365ac1e7ab3adbea9430789f83abf4c5
18426 F20101118_AABBHA interliggi_k_Page_129.jpg
e383e6aac8a24be22abace671ea2cd4b
9dfc62838c4c06c4e076f65bac47530d859aa812
6855 F20101118_AABBGM interliggi_k_Page_151thm.jpg
5cf841cbbd1eaaa859651c3fc5db6348
dcec0d7362627ff7e4bc9117020220361c184a74
48127 F20101118_AABCKD interliggi_k_Page_037.pro
8921bbe848427f56f8081a65cc7abda3
509fd4d6e3bd09c782049e45f243c1f258f71a21
29161 F20101118_AABCJP interliggi_k_Page_038.QC.jpg
236e65e6df1a50709c57736591f517f7
98c63677d4984e28543c22eb6ece279f7a50c4b1
364 F20101118_AABBFY interliggi_k_Page_130.txt
4cb585b2316f2c5f3ae4bfbe1307ca85
ea67f201341a8df93abd1efc5b436f58de42a5c7
F20101118_AABBHB interliggi_k_Page_089.jp2
f00d92c68d1c70f93d57e1e949f88394
75e49f32c3147def2bac62925fd8c2092616abf3
6425 F20101118_AABBGN interliggi_k_Page_014thm.jpg
5d5236363694417bbbb63d5fe6bc9909
58dbb5ca5cdfde9a2cb409f553393e5b89c1ccb1
1939 F20101118_AABCKE interliggi_k_Page_040.txt
86e27d4e045c526625bfc448ba4d9c30
8681d5b7af6e290a149d371f79f705687382c25b
41675 F20101118_AABCJQ interliggi_k_Page_116.pro
7056d0cfdc6d718f66288740476b7693
610784979d987eca1324f7df99d73cf3c7df6670
8653 F20101118_AABBFZ interliggi_k_Page_007.QC.jpg
6d67979c61239315ae7dcd750d9be18f
e3cd47c892f058678bfc21ce0acd024d79bfd5ea
2144 F20101118_AABBHC interliggi_k_Page_027.txt
f90f3dafbb01c3df7f763fab46cb2cc8
7a05ba4b3ba38a7dc6d58b319c3724051ca99723
20688 F20101118_AABBGO interliggi_k_Page_124.pro
25f7ecb3bba81d6b16bb22312521cfd8
39223b1473f96fdfd6c1675c49862252e4410287
78678 F20101118_AABCKF interliggi_k_Page_019.jpg
dbe0aa4615d3ad4916a851fd6dc53c36
6efecc673162db44df811df8cb23d08bd7a5521a
22945 F20101118_AABCJR interliggi_k_Page_127.pro
b0284d96e196f85633799900a9d8f33f
f5bc13cad5dd42146b357246fd28b4fc5845fb88
F20101118_AABBHD interliggi_k_Page_032.tif
3700b515b43b47f22dc3429f5a0fdbfe
903de6df7ddcdbbbf77ada462036307f1973aa8d
53543 F20101118_AABBGP interliggi_k_Page_022.pro
9fa11a8d65267231dec3f1fb5b2951ef
3aa3a301be8d0d9b09a52ce40698283ecf743a6e
23614 F20101118_AABCKG interliggi_k_Page_012.QC.jpg
d413b0365f406078b077af716934515a
b696ce8723411a7fde8b648dfeec822a2538cac6
F20101118_AABCJS interliggi_k_Page_031.tif
a3c45862ccf257e257c161bfd845174b
6a16457cfe2a4cb36669186c0a8350980e2263aa
7720 F20101118_AABBHE interliggi_k_Page_130.pro
b860fd8049f13a65762c354f80297b71
349dd66a579dd2c79e0ac741f13bd0664b53f387
1222 F20101118_AABBGQ interliggi_k_Page_128.txt
46da8d3cd35f531a8e46926382332ba4
368aeb3cf743583df7722fc2fdb16a366b02e7c0
10647 F20101118_AABCKH interliggi_k_Page_110.pro
ae2e8a7dba4833e05ba719f3189c98a8
1ccb7d353048c7cd2d433e6d8aa43ae17e9333c0
2082 F20101118_AABBHF interliggi_k_Page_087.txt
c47d339c33eb94ad51be5fccab4ae95f
ab3fd8ea04e25d9cf5cc17e83111885008ae9988
23787 F20101118_AABCKI interliggi_k_Page_006.QC.jpg
16d5f32b1b97255b27660e76b2e230ea
8d9753ef6c869cb75d11d95a3861d9573a144d76
F20101118_AABCJT interliggi_k_Page_030.QC.jpg
f771f83644abac15045409a159f0b806
9bf8f5528f33ee1f76f0d8bd6705f4aa4d53a474
34083 F20101118_AABBHG interliggi_k_Page_052.pro
8a8266585b15c2001be79f9f05dfce04
ac84a8d4857063b2c5edb60cfbf672ee22ea10c2
F20101118_AABBGR interliggi_k_Page_015.jp2
de01753bb79255979be214cfd5a3f7b8
c73ab2b825920dae469343ca8086ecad83beed0e
28353 F20101118_AABCKJ interliggi_k_Page_091.QC.jpg
8ad08cf854b7418e79dd22b33435144d
2edd4b7cd44ac85b8c542d41b11b1fd12be3220a
F20101118_AABCJU interliggi_k_Page_111.pro
93baaeb892062214fc8ccd6caf3cb03e
8eb5cc813c0ec7ef075d15e9f4ad6ef7c85eca56
13471 F20101118_AABBHH interliggi_k_Page_110.QC.jpg
cea4e637c2b469b59609002cd8d5d46c
92a38f47dabae1f41f66db8ed826cf57175991e6
28706 F20101118_AABBGS interliggi_k_Page_045.QC.jpg
84d0646b4b7255cf404bb88575c5ad01
2c0efc65d625ae026903c21138b1244e1bb62d70
31424 F20101118_AABCKK interliggi_k_Page_149.QC.jpg
a27468d1a4b891112e6c8fa867d12b3a
09679153ad6682828ae71bc364588cdeb33eb63d
F20101118_AABCJV interliggi_k_Page_051.jp2
40f5fb3e1a32f95078b115d4408a28c4
823c5556de59630ec264a88be5cd87e220e6fec5
54869 F20101118_AABBHI interliggi_k_Page_107.jpg
0152783b39f11fba45530203031dc975
e1783c19b2408a224fb2ae4d8fffda3c785fc582
F20101118_AABBGT interliggi_k_Page_083.tif
a498474786d832313ece15de7db1c1a8
66159cff50e00cd19b0e133bf84ebf068e336829
2080 F20101118_AABCKL interliggi_k_Page_017.txt
a7f5aa5a762e39d4e5bc95d4ffbcf18f
6c89e39d692e2663b4d4de9d751e8c15b7be0a47
F20101118_AABCJW interliggi_k_Page_140.tif
4adb927dbba6d5886b6cdec05366ddc8
899ad5b9cc4568e34c543d98aa3361a5e03a05d4
F20101118_AABBHJ interliggi_k_Page_049.tif
05557b4279b4546ea4a4a21533b38f41
b5ad2f97bf92a7d4d90d624b407eede2dbc4e30e
4950 F20101118_AABBGU interliggi_k_Page_082thm.jpg
0bd496a14ea82a0d682d2faee9929bf4
12beeb51fb59d9ee624c4ebc38f2e6b6853364f0
F20101118_AABCLA interliggi_k_Page_128.tif
90832ad6f3af53694042698a39ac539f
ea46a78dc7712e008a595a50679d322f8748a609
48415 F20101118_AABCKM interliggi_k_Page_140.pro
e0a5c170f06fb5ab96e7ae49e7e54ec9
f7371a62170c36cf2ddd727cbc6ed549fa961b59
18085 F20101118_AABCJX interliggi_k_Page_079.QC.jpg
e6dede743e88b23b28c10fa89fc2db86
d2164ebe0b2a7bf6fadd394e3794a4aba8662f0b
3645 F20101118_AABBHK interliggi_k_Page_048thm.jpg
9dd2e6c8321c8a9b3fa6f9c36bbf6ed3
7768eab864c2e712da1d56ed13822eb02cdaa346
6717 F20101118_AABBGV interliggi_k_Page_094thm.jpg
06a633abe0673206b899780c10547382
96b7f7ac232f3d8e5280a5ed506226c69be46608
65201 F20101118_AABCLB interliggi_k_Page_147.pro
a74f5f674620ae53d7826553fc92e83a
95387b85594116c5d273d6ff9750fb4d06ffc73e
83811 F20101118_AABCKN interliggi_k_Page_004.jpg
2ea3808e519cbe6b212e419bd7ff4c7c
d95f9c0a02d220d9857e732629329624eb18e120
16369 F20101118_AABCJY interliggi_k_Page_084.pro
cf386f8858722d10ff3cf9b815d2bbb0
c688aedc3a69b2e8dbbd60f14d4afdeaca7ca664
2125 F20101118_AABBHL interliggi_k_Page_007thm.jpg
a8800312e277f372fa2b262085e3473a
d713e00a417f6407dfee864e141d16a161551b36
69270 F20101118_AABBGW interliggi_k_Page_149.pro
3b98b5bda87ab195ecd432d85b1a3144
1de9a6f7ec33808d5e2dd54e774955565ece981f
2568 F20101118_AABCLC interliggi_k_Page_151.txt
d5beb964ea70e6ba899609291d96a9c3
2c323e540b3a8f946026a2fe794f00b855dd4fb1
6281 F20101118_AABCKO interliggi_k_Page_008thm.jpg
a264d19d15e035df4f36f34308325f15
16147287ca6626283bfa27cf890051c31ac81f83
11732 F20101118_AABCJZ interliggi_k_Page_125.QC.jpg
4c47d574893d6e40dc8dc4abe59ce2c7
16b8d4bc34ef2c66628987fe4702662957034cd0
12852 F20101118_AABBIA interliggi_k_Page_144.QC.jpg
05f38e31b6c210dda4c86790aa688884
b8b73821fce046ee108afea90d7d20e016acee1a
3909 F20101118_AABBHM interliggi_k_Page_003.pro
cba45cb4072871e8efff1ba5dcaaf29b
f9c974a234003cf8137c08fff98a1d59eeed7215
29110 F20101118_AABBGX interliggi_k_Page_027.QC.jpg
103dead9389f6a07faa425dbd9a232d4
4f6943cb9a6dbe3bb2bef9269a281f16fc8bfb7c
6698 F20101118_AABCLD interliggi_k_Page_027thm.jpg
b45f1e4060774d6fb80cf47d4f516e5c
15366c14a64aac67c5775f8d899bdada90778609
91389 F20101118_AABCKP interliggi_k_Page_033.jpg
94c01b43a8d7189f2046bf90a05715a7
d6f95391df13cf37921f46732c0a6a444c06c9db
6883 F20101118_AABBIB interliggi_k_Page_148thm.jpg
4ef4e5396b6278c0758c759979096ea9
d6670d0a39fca78501f95f3cf28518d764f0d9a9
25672 F20101118_AABBHN interliggi_k_Page_004.QC.jpg
0879eb52b1d580a49fb458b1d9d13176
ff28b1e7da881f1fcedeb799426849ccb94d4b03
881 F20101118_AABBGY interliggi_k_Page_105.txt
d4c0e62f1666c53ca95b1c73b9e3e38a
e6d7022a0b6f832a68e4b7ed97b2422c298fe8f0
2173 F20101118_AABCLE interliggi_k_Page_044.txt
a9dbb68ff6420eac59d2538d8c059432
87e788304268d147527e26abbccb54c6e4a84b9a
88554 F20101118_AABCKQ interliggi_k_Page_042.jpg
484402dd6af2989bb0b76f13d18586b2
7dd7d347d3bf51ede0fd9b2054c0742628746eea
F20101118_AABBIC interliggi_k_Page_036.jp2
ac6457996dde12a29ad832f67db6855a
7529dbb724a23afeebf540037fbf092f9bf73b23
24116 F20101118_AABBHO interliggi_k_Page_020.QC.jpg
35eb5f1c4068dc114826532c515908d6
47a66b3f97f531c5d36825e2b9ac9b69e6f484e4
6702 F20101118_AABBGZ interliggi_k_Page_129.QC.jpg
d0279bddda5a066eed475dbeb712a85c
4e6005dd6db08ea2d0e701732fe4ea511af346e3
17560 F20101118_AABCLF interliggi_k_Page_064.QC.jpg
0815e7b0f10f80a80ab69932f7f9d19f
73da5150cfea39f6a1c13a5d488dcbe606c61afc
F20101118_AABCKR interliggi_k_Page_099.tif
83e35a78e3df908364bd8a3ec30f5cfa
ec309930c49713a2da97edb1557f6693decc47e1
6074 F20101118_AABBID interliggi_k_Page_030thm.jpg
75154644153522c5dc8c6970b09b014a
37d0c6a9ae4a10d012134067e1a9b8535a9fb6e2
1051940 F20101118_AABBHP interliggi_k_Page_023.jp2
6fbb1fc16650bfade1e4ce06c26bfd54
8899c8bf2547a8666b8a96eef5d3b32f59b26724
88265 F20101118_AABCLG interliggi_k_Page_024.jpg
c98536795c72ae0056ba0701e69d9c02
f0617df2e9795551ea353c6cfa8d468eba4b5319
F20101118_AABCKS interliggi_k_Page_119.tif
24945168a37809c3df3c6bebd2471952
e598a6cf9989ba13758d805c8e6fc118bcab32b1
6748 F20101118_AABBIE interliggi_k_Page_134thm.jpg
59699948df9a1df5af64253c79811c58
cea1ae963da057998f00b1b9e11b8d1a0e0be6e1
96 F20101118_AABBHQ interliggi_k_Page_002.txt
050effea9ef179aaa88f5af6eb9cea1c
fb12831ebc1b9c0ee8901801787bed9854761e69
F20101118_AABCLH interliggi_k_Page_134.jp2
797ed7a980bb67fae8a510a15226afd9
11fa64fb1c7ad66e1e69301c6b3810177a0333b4
F20101118_AABCKT interliggi_k_Page_014.tif
3f2e52ae5cae6ff59c0c2413958f22f2
e6c1bf6fe9cb3aca075cc838fb8b146e3c422bd4
1051977 F20101118_AABBIF interliggi_k_Page_045.jp2
a8c97fce3ee4bf8b46cf60774006146d
ac279bec9bbec29bbc5cfa000c489f6133bce695
473 F20101118_AABBHR interliggi_k_Page_060.txt
fff7a6d077cb79098d90c37762dcf9fa
c5eef90e2575c553c5f51c4ab50c61c24a8e37b1
75430 F20101118_AABCLI interliggi_k_Page_140.jpg
a072799fdc5ac4bee5c4221e87163bf1
d6410bd2331470d26831e9814ba44643903ef43f
2081 F20101118_AABBIG interliggi_k_Page_021.txt
d17d7d3a5b30b75a4e7d7b31ee535857
45d9b64458221143259e1a90080452ff9757b499
2046 F20101118_AABCLJ interliggi_k_Page_041.txt
84a1c1495cb5e8c5df215ec2227403df
16d3f684f3e6b5512c23ec9d53da5dc6c2143563
93554 F20101118_AABCKU interliggi_k_Page_008.jpg
78bd36ebe62c750ad8b29b2e57970db7
a910630ec5e568ae3531afdcbd4e93e85c3552ef
84282 F20101118_AABBIH interliggi_k_Page_118.jpg
6f8d8d834ce258c5714288129685ed79
1cf1cab6637c7efbb5848228b23f934cd05c3b72
80983 F20101118_AABBHS interliggi_k_Page_067.jpg
741acd491c58a7af5f97b9f01caed9c6
1e24e9805b791de6a1efb22de6a46cd1ba28ce16
46548 F20101118_AABCLK interliggi_k_Page_122.jpg
960215ba68f36e7cec88f644785a7990
ec5fd9d93b2c2a3c61e6baf1ae2a851cf25a8b11
772 F20101118_AABCKV interliggi_k_Page_003thm.jpg
5b30dff5ddc2b6ea8eb1bcd0430bf57a
e1f497b5a9dd03ebb0f27850d2a8fc56abf65437
55364 F20101118_AABBII interliggi_k_Page_072.pro
0acc269b864e6f1b523ef696a3847e71
e03e41b6d5631de7e286f256494bf83f374d75c3
26739 F20101118_AABBHT interliggi_k_Page_088.QC.jpg
98863bddcd0a18b91f52967ef505dc3f
c724d16e7603fd42051fe328bc443fb1fb37d89d
15465 F20101118_AABCLL interliggi_k_Page_053.jpg
f70da7a2ab6bf831900194426d643408
f7a1c6078d6f2908f84224b6e6b407d10f75ce20
83832 F20101118_AABCKW interliggi_k_Page_028.jpg
bf2e79557d917caf37cc7e484256e818
d58d81f875f9211f969a54dde9748504b9bf9ef4
F20101118_AABBIJ interliggi_k_Page_098.tif
7666ff312950726d1e087414506ce886
8a1642943ea6fcb1e2684b139aef4b01a09ffddb
2253 F20101118_AABBHU interliggi_k_Page_130thm.jpg
30407827ddef7f289506e84b996821e7
7260f634277640de01dd4f93fd97bfad1dbc3650
2620 F20101118_AABCMA interliggi_k_Page_147.txt
d393a635772e6fe175f3437b64950665
7352e96d28d25192c6d80b42cb23946824729f8e
13054 F20101118_AABCLM interliggi_k_Page_126.pro
2fbb1c5789f8ebc16fc1c172a9988d05
bd6125b2f92bd4ae71c7ca2cda5c5e1cb41bcffb
1051964 F20101118_AABCKX interliggi_k_Page_066.jp2
93df66ad02903a74bdfba3b720dd8791
d2f692a33e6718199c252d174a447e2fd3f29897
704581 F20101118_AABBIK interliggi_k_Page_110.jp2
3794f05e2faeebf4f527ea89876f359b
005bf245cf0cc6588ff4f810e92f7eecf0a6c2b4
2143 F20101118_AABBHV interliggi_k_Page_022.txt
e4e4d8e0ec5c517acf4a27bef18c09ea
81799e1b733fa940b1cd24616c0a7ebd2265e801
4173 F20101118_AABCMB interliggi_k_Page_127thm.jpg
69078ec6e120b634d977a22d3b72b775
4bf14b2a604d3fa1a57f836e24ed58545d2549a5
779 F20101118_AABCLN interliggi_k_Page_076.txt
4402182d39d643f7bf89b1a7a4b805c9
aa359913247924e71842f25b282941e96f2e59b6
522471 F20101118_AABCKY interliggi_k_Page_112.jp2
305cd194bf9fc95ddc9c346eb7ec6d6c
6dde41a3f5f9f3218fc550588bf712aa314024c4
134 F20101118_AABBIL interliggi_k_Page_139.txt
21f1809d653c75da0ff74a702b0ba81e
013a92f47b68f4a00e5bc79224dbdcfb62025204
11970 F20101118_AABBHW interliggi_k_Page_078.pro
5600e3408cc423dc30403acded309c0d
dfb8a91416b72c316a2f2adcfa2c2c26a956bda8
30292 F20101118_AABCMC interliggi_k_Page_062.jpg
f3ef3e856afe41e95e4df980562d23db
9e8a45de8ac0e457947df1edfd5236c824156b88
15619 F20101118_AABCLO interliggi_k_Page_075.QC.jpg
0378e54cd752840ea56a675909945a54
84cd30a31170f3bcdbdaa58fdba91607fd2db307
88409 F20101118_AABCKZ interliggi_k_Page_016.jpg
0a9e4303074689f25fd65033cf188909
4afc89f3bcc3e42180ab397a49026d5ad95e2961
6471 F20101118_AABBIM interliggi_k_Page_041thm.jpg
3bbc115070b2166215ffa0b5a48d97c3
5478c3f7c0003d5a993c02e9a5fdb6726a8b7cec
18369 F20101118_AABBHX interliggi_k_Page_143.QC.jpg
e5dd1ad4d50720f2125ad297c331e3be
1210e5ae0237773f8ab03d7585c95fb5e947bb67
89051 F20101118_AABBJA interliggi_k_Page_090.jpg
7126fbf93ddae7bc0a3ba45973fef068
2f47d84a6d13c8f351f001baacf699142ab61d88
53460 F20101118_AABCMD interliggi_k_Page_024.pro
48fb8134502432786cd35d47954aa4fa
dd10456497b47144c2eac63a6ca6e5218b36d74c
27016 F20101118_AABCLP interliggi_k_Page_133.QC.jpg
6e9016bd9575e83cd0cc60588b3c7fb6
6798f945a9f8c6146cde238734cf298242a94e0f
6603 F20101118_AABBIN interliggi_k_Page_115thm.jpg
89cd783d414c64617d041f6c13745535
e1368825e6e527eba10bc535e2ea8e21f65284cc
679 F20101118_AABBHY interliggi_k_Page_078.txt
a69e003776595a58d701c7caeb6486c4
a889cedf92969f675178444150b7a957c45af18c
F20101118_AABBJB interliggi_k_Page_082.tif
94a7c5a65f4942c018635f1d73590357
a0da6224b7383008155f190513984757ea39174c
24451 F20101118_AABCME interliggi_k_Page_063.QC.jpg
936b0b7a8dda052cb3e90f809950ab5d
8dc8599bf8c275b4afb77d8a14a1542deceb9f22
11495 F20101118_AABCLQ interliggi_k_Page_058.QC.jpg
a0db232b07d4d04e529ba6678d5347d9
835f27edbc5498c86465fdafc584276828322ec7
20084 F20101118_AABBIO interliggi_k_Page_051.QC.jpg
d8e7c21b86a203a5fc17092d9662ab3c
510d06690826dc62845fe1602f8d4b6b17279433
F20101118_AABBHZ interliggi_k_Page_007.tif
62a82f310c1fb27fc4b24f8648216f21
09ffaac4609fa2e0ad343dc8bc791de457a67e17
64357 F20101118_AABBJC interliggi_k_Page_052.jpg
ba42af78ba3640bbf7daa934f0cb432b
206f9aba749089b3e3d9bb0e29499b5d26975162
F20101118_AABCMF interliggi_k_Page_033.tif
8a413e22f80f1dc560db89b66a88b8d4
0c43e4c3854d381d63ca8be834182cc666d6f27d
55519 F20101118_AABCLR interliggi_k_Page_010.jpg
7db62c38af328486b76a37dce405afd4
66b85b8ee5afe2c8d90afdeac20d89e4f7cf5601
1394 F20101118_AABBIP interliggi_k_Page_082.txt
f2c348c49d141dbeab565c9c5bba7b75
19b2d79bed9ff4e6ad7e9baf985ff95766382153
2122 F20101118_AABBJD interliggi_k_Page_070.txt
2f49db1250a4f3a352b8f756b0e00d30
d6462ff461fdd73bb4105294dfff7c2a93177936
28999 F20101118_AABCMG interliggi_k_Page_018.QC.jpg
c28e61e0bf5ed06a7e6a7a1e931faef3
5e040a6081b4ac0fbb7632334c9c0d804fac4876
101383 F20101118_AABCLS interliggi_k_Page_145.jpg
99c401e4ffa01bc843b56be6ebe2e80c
3b000dfa3f30329d8fb156877be00a0ed9b5190d
29109 F20101118_AABBIQ interliggi_k_Page_093.QC.jpg
b5c1ed50f718f9f0472e2faaef57b3fa
3c57a2aa0af69f26e2c3c5bb175a26f1339f5e3a
3850 F20101118_AABBJE interliggi_k_Page_080thm.jpg
110958873c395675102ec0293194af21
9118de6b534915813f2f5c6960aeda436a9f9cd2
6809 F20101118_AABCMH interliggi_k_Page_145thm.jpg
29f7071aff8d650a786bed46d3d29bc6
2e515dccc068c8b7e6f9b30d24d99da1175adf34
F20101118_AABCLT interliggi_k_Page_027.tif
922917fca8cb9359a26e15c7f6e47370
719c442ce643c86b7f0fe25674b52ed78589b43f
25229 F20101118_AABBIR interliggi_k_Page_086.QC.jpg
b6945e42eb8466d674051c8663b5dac1
b2c5939be91a9294a30f3aab0952d52fbdc582d9
F20101118_AABBJF interliggi_k_Page_066.txt
8eb5e7fde791c911e2932250ad18156e
65492adb1bf3eed56e71a3938eb79706ec4fc107
F20101118_AABCMI interliggi_k_Page_028.tif
a6aec0fe8eaa37f65bc9509f4f6133d6
b9b13dd23dafe54c26b8fc03401e8ab0293bb8e9
6329 F20101118_AABCLU interliggi_k_Page_088thm.jpg
f79171ce33bd407882b4e2609ed9095e
22deaf1d81300df4e190f4b036b838e50f5a5be2
F20101118_AABBIS interliggi_k_Page_108.tif
5894df314383eb04bd95ee0ef27d0209
2f7f9874276aa2b3c9da882030a2f266e03b6e8a
6215 F20101118_AABBJG interliggi_k_Page_029thm.jpg
df17d02dcbf3aca26f3b7cb187692f35
12be190acfdcf7563f48f23e0337d08df95b5202
F20101118_AABCMJ interliggi_k_Page_136.tif
950fb30fa70b74ba698ff541f6e75783
0ea530337af5299573940ea4e6becd4c03af016e
6160 F20101118_AABBJH interliggi_k_Page_069thm.jpg
c9aedc4dc7e3c7afe007a2fe10c1f0e7
cf78abf08e807c7049a9b43a5cbfec2422e61cd4
14893 F20101118_AABCMK interliggi_k_Page_122.QC.jpg
7dc464c608e2dfa3af8f5dde603eda8f
f6efd77c396d1fc018d23673ec6d7a9ee18ebfe1
38145 F20101118_AABCLV interliggi_k_Page_077.jpg
7c7732f54a98fc20a5d916cb37253623
9975d847a91b4ce2f7d6ee9287b1b9efab7f135d
12326 F20101118_AABBIT interliggi_k_Page_102.pro
83ee9bb28f5b39b13e5e7c7ba0c74796
cfc6dbb5b4526cc4fdf2d94433ccd7b45c7155be
47032 F20101118_AABBJI interliggi_k_Page_108.jpg
a03d55aa31f1d5564f4083d21c736674
79ecaa23f25811deb7a09854adfedaad4d0dc22d
F20101118_AABCML interliggi_k_Page_147.tif
2781ea1504b8feafb61e15b0fb9a28f6
ddea9beae1b480b5f9fbc344af8c22a7b8e67057
88739 F20101118_AABCLW interliggi_k_Page_036.jpg
7fb85841aa6ac1ec3e5aa371a8b1ed03
f370d64e0dbb1fe695ee8fba230ecadcc43e1f67
88411 F20101118_AABBIU interliggi_k_Page_021.jpg
e9e4459e8949678a51dc73930bbca98b
1eb5d097748573553a3d22d8f4c5a9aebe625b7f
3185 F20101118_AABBJJ interliggi_k_Page_078thm.jpg
4c70f04a768471a52ec5bf87b2912334
41fe69d2fed78bb15ffeba6b19ff759156bda121
1999 F20101118_AABCNA interliggi_k_Page_034.txt
85e21bb20fcd204674005270a2d8ae50
6e592eb9eac861639635d778959dcbffd952cb2a
14099 F20101118_AABCMM interliggi_k_Page_112.pro
016c8f8871866ec3f379c84d28288c9b
37a4cc8fef8ca7b168860771475c65840a22444c
F20101118_AABCLX interliggi_k_Page_097.jp2
c4f1733f6b576437045da9d133d6d690
14ecc9378c09fb7ec7a06c44d50ff04a943ea443
1233 F20101118_AABBIV interliggi_k_Page_075.txt
424d0f0dfb47e4f23de3f459ec3cfa72
0e1ab98fcd13ff0151c6a145f39d38d4aeea0b03
845 F20101118_AABBJK interliggi_k_Page_100.txt
11e63aa9029b3a3487f86f7ebdf1d430
b219733ebbae744ba01edacf04513867c6b56e24
9540 F20101118_AABCNB interliggi_k_Page_062.QC.jpg
db93979466d7a80a07d923cc9de51161
265443339b1aa0f2ba2cb3c06008eb99cf0b5071
2070 F20101118_AABCMN interliggi_k_Page_065.txt
296d2c9ad53e3af0c56250ef39df122a
c26f7322e6b4ec80935535e37380330b15b4991a
2174 F20101118_AABCLY interliggi_k_Page_098.txt
c22eaff11ab333e06b912d078bc8af4f
060ee36040dd1178e3777d84457f2355febd8521
F20101118_AABBIW interliggi_k_Page_041.jp2
91a5fe237d7b35d95d4ac8d20ee4bb96
1319e3a644d33956515596ffbca41d5ad5de560f
49129 F20101118_AABBJL interliggi_k_Page_067.pro
c0ade382243f34d92618467ae187b083
1f29106c25f742d0762a9d4a166da0456420b3c7
6245 F20101118_AABCNC interliggi_k_Page_073thm.jpg
8c01ed29a29aed02e76adac819f1ead0
447012c1e79b49ea5b8226e57aa91fc1f0815f8f
2884 F20101118_AABCMO interliggi_k_Page_008.txt
cb20b89045d4b0b85cfaec8bc933c592
94ef5e85920cd3fc952d515efa5c577051fc3952
14005 F20101118_AABCLZ interliggi_k_Page_013.QC.jpg
075dbb5e5334c0e4af28f3fd3267dd7d
fafdad1e5bac7eb3b73c3984121c8313b79349f6
2085 F20101118_AABBIX interliggi_k_Page_089.txt
aba1d615f3116f83da6930a839c64639
3b0ad7b25d125db8eafd3031817278cf85b7163f
27243 F20101118_AABBKA interliggi_k_Page_061.jpg
a69b327449ef1d0aa7b38688908f6c4f
b7280d2bc06a547b67da96423518875c818e83b8
1051923 F20101118_AABBJM interliggi_k_Page_028.jp2
46e3223fe0d4097cfe9b3d8ca10bfef3
cd3f93fcb04d89d1fbfd6cf749176fa6b3903abb
6576 F20101118_AABCND interliggi_k_Page_016thm.jpg
fe518ef689e5e4394e132dc1c867f202
4085057a3f54320b76f5592c9e6634a22ac2973d
27459 F20101118_AABCMP interliggi_k_Page_120.QC.jpg
90ea700768f5da998c4b368983f830c3
2d182ed55ccd327efc450041b36c65e479adef7a
2112 F20101118_AABBIY interliggi_k_Page_024.txt
d6e79d2b51c07574fbfe1e5eb78db5bb
3d9e88cf257f52f3636d2263972aff53929a29f6
F20101118_AABBKB interliggi_k_Page_096.jp2
248d15ad81c74cf65eb363979584e92d
63f830aa6b414e63467f631d51c74b8317e5893b
1355 F20101118_AABBJN interliggi_k_Page_051.txt
2b2601fc6628bf74c406eefef3cee651
caa1bcc3e01917559d2392ab3356d2406369c9ae
7039 F20101118_AABCNE interliggi_k_Page_147thm.jpg
67006c9e75944fc5ae7b4b73c02c96d5
1c8bfdb1b7cb3ecb75dd30c676a64c7ec5cb1f42
F20101118_AABCMQ interliggi_k_Page_026.jp2
e08ed8c80255ec38a16c088785aa4ff5
b3e25fd2849b8602f9ee3e8bf20ac06ef185567d
15767 F20101118_AABBIZ interliggi_k_Page_061.pro
e793e3bcde2b0575262796de82c07471
393bdd33765dafc3aa32c2be55b0dc6ab24874b5
F20101118_AABBKC interliggi_k_Page_125.tif
d0a447516b47ff2b6851b23ab0f8cb07
6bfbcb93898008f02eca7796b3a301f3a8681613
F20101118_AABBJO interliggi_k_Page_069.jp2
aeb5480a4420dc97f943d327534650df
97ece077e89ab2102908575dd3e5f3b07c1a7cbb
11904 F20101118_AABCNF interliggi_k_Page_102.QC.jpg
873c56267a1424f2356e5ded47ea8fad
8094d1d355135930ab34bbe9586a887f48f37e77
36474 F20101118_AABCMR interliggi_k_Page_010.pro
aa9cd264e005280b0ef1d2cacc0721da
9bd180e909242e475b78e054d549253415550a55
1940 F20101118_AABBKD interliggi_k_Page_023.txt
32980b987507060ec767d0aee502cea7
f0da7449768bf43231a44edddb8469fc61d15d1e
50207 F20101118_AABBJP interliggi_k_Page_065.pro
03ffc578dad5413f2ec4364869d56876
f4807c3c4bb84910fdd864f82b1a0892db4f8958
79474 F20101118_AABCNG interliggi_k_Page_142.jpg
400d99cf41f175dadaf156e035cd8046
6c850fa341aaaf4a076d1246a4bf752418957d66
2123 F20101118_AABCMS interliggi_k_Page_061thm.jpg
0898d759a8f9e8fa42d72c51ba525c1c
b3789bcf4b379551ba3c1ab4eb520c67db623a20
F20101118_AABBKE interliggi_k_Page_102.tif
af33bce01789a3d9b2b972498adcaa09
ec96bdddd5449a07d266a6d0271647a3e021aefa
106581 F20101118_AABBJQ interliggi_k_Page_146.jpg
2645bd25790f8d35860a59444371e8e2
0a5ebea4f27d2a4464d1f06d7b8e74d0c5bb0446
17200 F20101118_AABCNH interliggi_k_Page_055.QC.jpg
c0c604a182c17c126c1607d6d3873251
3e3c7a0e123e1c1c50686b6ff3285fa5b1dcd940
F20101118_AABCMT interliggi_k_Page_062.txt
a9b88fe908383fd36a03387e3ff4e880
fba77c1797db4e5c4841aed7aa107f39da19d4c4
89560 F20101118_AABBKF interliggi_k_Page_121.jpg
4d4f89279fc7e435de94289e856e3f87
b224a2a0fcacea385662dc2382751f612116de74
26704 F20101118_AABBJR interliggi_k_Page_041.QC.jpg
e77dc71c514fe054fcdc4a6e44e4583e
d2c8ee5f9bdc2394a526fd5dbe5f01e98614449e
28441 F20101118_AABCNI interliggi_k_Page_111.jpg
a3ff3e6214c4c9be5a35eaef3644984d
0c1d31c4d5ad9e208991135406ce84b842ed139e
F20101118_AABCMU interliggi_k_Page_109thm.jpg
43aef0df06dc83fc9179ef5fe7ad9b66
8cdc270b15ed0af2e9501fe7e0eab47c5dc7dd73
3624 F20101118_AABBKG interliggi_k_Page_112thm.jpg
bfe6b0e492bce664a162cb2e58383cd7
72741522b79a2a80cc356662bc2262f2c243b123
89068 F20101118_AABBJS interliggi_k_Page_098.jpg
ce2456be8576e64e0b59aa4fd542c64f
b63d234535c1705a28ed2572a919b2197a702cae
4847 F20101118_AABCNJ interliggi_k_Page_085thm.jpg
b197e6bda90c880f78089226348f6271
f2efb17f0b81192928fbe04eb5b69e6081f1656c
84802 F20101118_AABCMV interliggi_k_Page_088.jpg
8f327a55536c32ba2417fbfc1aab39de
4723aa8f6dae112c5920ae1b95ddc16d122ae597
F20101118_AABBKH interliggi_k_Page_149.tif
df3031d779b527ee0088c3317dc6738c
f19ef08b3e5d95ec085a15ce7dd9ff680b5dfa4c
F20101118_AABBJT interliggi_k_Page_012.tif
67cb4e6406c6368c9007acdf8388a3f7
0a06173e32c28b85c044faa5181eb0f265678941
12814 F20101118_AABCNK interliggi_k_Page_100.QC.jpg
0c31f97607791c2937bd3b6224572071
573ee0509d4069b6e115d77676b022106ba2fe5b
22326 F20101118_AABBKI interliggi_k_Page_050.QC.jpg
013706b96725a43956fd99466c3f26a5
33511b28254a987486b9eb46ddf49786447857e8
774 F20101118_AABCNL interliggi_k_Page_131.txt
b6b301469864591ee722da902caf43f3
9c49b2905b263c99399cea67d38618ff06036768
1353 F20101118_AABCMW interliggi_k_Page_053.pro
04f159cedbf49a8ccf299351ad473b37
4622172ed97fce17dfe2755e52112d234d1b8dc7
F20101118_AABBKJ interliggi_k_Page_037.jp2
eb9d998054df0b79189867ec4c68808f
1c6d2e454eebfdd648866a84b780a0a71416d0ce
70065 F20101118_AABBJU interliggi_k_Page_099.jpg
0f17afd2a9b1e19a106511f5d9376a71
09966dd11998bf1bb698af1a7f216a0a00f746bd
2768 F20101118_AABCOA interliggi_k_Page_139.QC.jpg
4965940ab4089410a8ea81611587de36
cdaa576abaa53ec35549c12165c5f2b1be9d5bf2
654110 F20101118_AABCNM interliggi_k_Page_104.jp2
722f41d2490cec3d2d69a61eb48d3de5
29232e76a513acff95440f7598a7b1dbd0123dc7
28426 F20101118_AABCMX interliggi_k_Page_090.QC.jpg
8bb32577a158d59671b401385d34fd4c
f3353674635663a7e6e86363985325bd345d8b3a
23707 F20101118_AABBKK interliggi_k_Page_076.jpg
ea5b2fb5e9f40df9da20ac79de14d76b
5d8bc7365326a81c18b545fa67469c1b9c1401c8
91294 F20101118_AABBJV interliggi_k_Page_044.jpg
726ad2ccf1b9ba4f485a251a34e9bc76
a26129c1d89da89f417ea5356a43dea0fdd1a1a7
F20101118_AABCOB interliggi_k_Page_117.jp2
f7b30c62f31772f81972c406429b91bd
bee292d58cf1610506edd28a845c80a4d7348667
F20101118_AABCNN interliggi_k_Page_150.tif
89100f855b43660547612627524187da
8e4222aefd6445a4fc47bb3404c844c724a91619
F20101118_AABCMY interliggi_k_Page_004.jp2
0d8705113d7c32408d3fbb2fd3506163
740354c97129f6f5f12074328f1413a68c9b32de
5966 F20101118_AABBKL interliggi_k_Page_040thm.jpg
e0c4dc75389879ace8be2f4d7a727825
3a6f7ce33dc3d7bb09baf24279faa522afa43a9c
2519 F20101118_AABBJW interliggi_k_Page_106thm.jpg
e81888de67589fde2439946b3959cc6e
e6f4c1363d94bc9a212c5b4c6241614ed40d6b3f
6413 F20101118_AABCOC interliggi_k_Page_114thm.jpg
fcf3b8bfc0fab2eb50bd083145aec1ec
90503d80116435ec19fce3a9ebeea51695c1273a
25250 F20101118_AABCNO interliggi_k_Page_023.QC.jpg
5583917655ddfc35cca19cb101978899
770657ce9c21c76868dfc547da86ec889bf4e5d3
F20101118_AABCMZ interliggi_k_Page_134.tif
5c2ea60a025d509c25361dd7d1367296
c21f6824090692b5b6f7852d681da2888712eba7
250646 F20101118_AABBLA interliggi_k_Page_129.jp2
e20d76a803d0ea32f25bd5cda4208035
fe258a791393e3e3a09ccb1e4caeae731b872ab7
2365 F20101118_AABBKM interliggi_k_Page_143.txt
b9f628247e52fd5ddf63650f041444b8
dabc869a12f1600a44b0806d8eb2ebde84022acb
55488 F20101118_AABBJX interliggi_k_Page_091.pro
96c203d122794f26591e13756daae0ac
9093e5cdc20eb35da922f9f45efe01fbc9f98fec
7062 F20101118_AABCOD interliggi_k_Page_150thm.jpg
4bc13526ed09dce9ffe92406c50dc364
47891d8c104972473b86047fd55550baa9d9cfe8
8179 F20101118_AABCNP interliggi_k_Page_106.QC.jpg
642dfbdbd4509496007aff7223fb88b6
6b239806a3377ccd6d8a0508d748422d0f411fb7
3901 F20101118_AABBLB interliggi_k_Page_002.jpg
94b9375e4725da51cec79aa92ae62a32
f2d418ae7861649cbbeb69388a183d81abc8da50
1395 F20101118_AABBKN interliggi_k_Page_144.txt
3d7e8f250aff95905b0fec73dfa3b9fa
c8fcdfe36e9254cb5eea51cacc360ab480a1b080
23599 F20101118_AABBJY interliggi_k_Page_001.jpg
65c245506cee08664811254d5a998bf6
3700bf4e1e276dc94033bb527d89401f42588f3e
1340 F20101118_AABCOE interliggi_k_Page_108.txt
40bd3cb325b72a9b64ae1eddc31e3c5b
8406327910e0275b9c3c9f7243340e9d939b2bd5
F20101118_AABCNQ interliggi_k_Page_003.tif
52901b3974b6e35eee9958ee9215f32f
5024fd2d47f0134b58a7508c80ff20f293268e8a
F20101118_AABBLC interliggi_k_Page_096.tif
9e14c29a944063608b29c1e39bcd5cad
baed7bb73db7402d93154038e4fe2f102c23abdb
F20101118_AABBKO interliggi_k_Page_124.tif
8db986d0d7685038495019ce8c9ef0a6
c9b554c682fb074decffb3244e9476bfde348ea5
F20101118_AABBJZ interliggi_k_Page_022thm.jpg
90995b50f4a8c7e7e57f56112a6c5399
3ebe28bc718100cc231656c3013d3143d3709113
30002 F20101118_AABCOF interliggi_k_Page_150.QC.jpg
7bc29a4e81460957734dc39d8029aa2c
2f4fe3f03a70479f1f1f2ef1c81517e0c4e72906
F20101118_AABCNR interliggi_k_Page_031.txt
66234fe450ab587d740d51e38bf11b7f
9d11dabae7f0a58be519ccce1f8ab1333953f174
3584 F20101118_AABBLD interliggi_k_Page_108thm.jpg
41627f25fd06b0861b32e88ddc47fd12
7060dd49a6399c70631b6cdaf2477566f724e38c
3801 F20101118_AABBKP interliggi_k_Page_010thm.jpg
7d6ad2540e800719238cd3f0936439c3
9e388fc7da9720116ef8f8f319d14162956ae83e
2977 F20101118_AABCOG interliggi_k_Page_081thm.jpg
33e24afc194fbff88ea0a4387c575f30
aa0ed41f17d33252a088ae7314bca929ac61d6d0
F20101118_AABCNS interliggi_k_Page_070.tif
91343781dff777b12f60d80451f034f2
71b0f873c340c9fda5e9ecbed31af523911584a5
6310 F20101118_AABBLE interliggi_k_Page_119thm.jpg
234dc11f33d779060155c7e065c67c83
5494360977cef183d7fbe6306569e0247427f9cc
17332 F20101118_AABBKQ interliggi_k_Page_057.QC.jpg
64fc12b5998303225c40b026750801e1
01ec7a8a709cfab70b1d3628f0ec2a1921198b69
29918 F20101118_AABCOH interliggi_k_Page_009.QC.jpg
8b1c1773eddef43b697cf64a3bcfb9b9
0d46ab7881c75dc94d6911a7ec817950602b6ef9
28079 F20101118_AABCNT interliggi_k_Page_026.QC.jpg
cba9e3723301b82e54ed2179d2be4546
a724ca3904274886542e0afd7dcd5673368b251a
1051936 F20101118_AABBLF interliggi_k_Page_091.jp2
6f24463cc63b01a55a2d57bb1bc81a13
2578550a3d891f32be58c3d70973ac2ac8c8be9f
5371 F20101118_AABBKR interliggi_k_Page_129.pro
5700ce936f00aa4e0e7567687edf4302
45be29c632009a7dcceb4aba135e84a7e6ff216b
26074 F20101118_AABCOI interliggi_k_Page_025.QC.jpg
b68ea42c852ac88204aa23551ef57fc0
c777bc7ec7640d5e3829d66d4def49ddf885f8a3
F20101118_AABCNU interliggi_k_Page_035.tif
71041e0a3bf4e465c9c5dfcd0c8ec823
922da03b02ee0b5c0ebae9379ab68f31d9c4053a
55670 F20101118_AABBLG interliggi_k_Page_018.pro
f679d4abfab1346b489730715d5c6fd6
ec2522355785245cb8cbcd65ed1c9cbb0ae7eb50
F20101118_AABBKS interliggi_k_Page_107.tif
9e1310b0a5e5f13c8d7630e45d1973eb
a5fa8f5f5146b3196968b76f78cbb08cd9009edd
2075 F20101118_AABCOJ interliggi_k_Page_115.txt
4504f8b367b2e025e081b0c0c5e1fc52
e275a650d2823e14a34449efe9842487f1ab20f0
88897 F20101118_AABCNV interliggi_k_Page_135.jpg
04b77ba12dba79d3ccd75981a3c0ded0
cac1c98a3bb52fde632985d5a7a19297a096db5d
2193 F20101118_AABBLH interliggi_k_Page_018.txt
a41c93e1fea364e0e4322b40f4ac0b63
9b6ed1ccc75b6ea884e7212d701833cb11e82e44
29459 F20101118_AABBKT interliggi_k_Page_151.QC.jpg
f1641e5e5a15c471fac813c9f86db293
ee2df6303403819e2b266f2c44cb2b76cfad2893
1968 F20101118_AABCOK interliggi_k_Page_073.txt
8d439914797decb0ab441a8458625add
500dbbc193ddc822833c612d8635d108bcc3b1c1
83931 F20101118_AABCNW interliggi_k_Page_114.jpg
356621f8033c0d120887948541b8535a
388f6c801822e4c84c900f059e519167cc49d619
70387 F20101118_AABBLI interliggi_k_Page_049.jpg
84fafe153ac217738899632648aabf50
79946ba49edbbf0a1785f7a283f4030105cec50b
F20101118_AABBKU interliggi_k_Page_078.tif
6d7cae49d5981b726d555ee3b529b2e7
6fe01d5d22ffddb3a011730c10e085d1a0e60ff7
30658 F20101118_AABCOL interliggi_k_Page_054.jpg
0b84929b4db45bfa5258f903ddd45b57
5c6f63434206e5b1823b50180e8b56a9eeab71a6
12874 F20101118_AABBLJ interliggi_k_Page_080.QC.jpg
e99bc503c3c7f5458fb76e4b5c8d6e70
225a38681730f88952bdf83cf9ccc7f2d325481a
57705 F20101118_AABCPA interliggi_k_Page_057.jpg
c46d9c1201f7d774e45b18c1ee3d2f4a
2309de0a6d145c4c76a68b0da5ec887b33be9cbb
1982 F20101118_AABCOM interliggi_k_Page_004.txt
85a516aa2fe3c4d226d95d86a2df8553
c966dd7fc71671881e8bb3b5fc3c5f4a6c1400e1
1051891 F20101118_AABCNX interliggi_k_Page_137.jp2
0c0d77084f8751df37d5ba71d28afcb8
e96b5ddee02d8672dc5a50d59fd5caa606999765
91160 F20101118_AABBKV interliggi_k_Page_045.jpg
231e6b6db53b0d28a568c3fe297366b5
be4ca7a919b21e2ebc28472daf1f0bfc81ac7680
533 F20101118_AABBLK interliggi_k_Page_154.txt
801a4f8f721f9f468eae599ce5070983
ed03ebfa9a399f1f81b3ebf4ab5915fe7f768e16
91716 F20101118_AABCPB interliggi_k_Page_059.jpg
b30333d45561c214ff5cba090ca058a8
42077c58c811dcee884bf82b4f024d1c92c9b5f2
511 F20101118_AABCON interliggi_k_Page_001.txt
89f56dc3dcfe609fd40e8e9912802bdd
9fc02c4ce0dc18df80dfb4d6665cfa5ef539d8b7
6565 F20101118_AABCNY interliggi_k_Page_118thm.jpg
24694a15fe2bbf634bab4b915f49c1fe
b57396b2d20aa7c7773cdc4c1372f5f6f1139dc4
790308 F20101118_AABBKW interliggi_k_Page_079.jp2
f8216d1822016675976979d8f431648c
6e7e78cf817e561e3b1df3b5fb8890014e9bf40c
28085 F20101118_AABBLL interliggi_k_Page_015.QC.jpg
8edeff0de870d377c5e1bfd1b2bc1c68
264e43e5c87f0903c2824505c404ab9c3d9a956f
55066 F20101118_AABCPC interliggi_k_Page_064.jpg
c79ec59c36d810e5d4a582b12e26916a
643a61a9cf13bb2e09789843b7566bc9c1c4fea3
1731 F20101118_AABCOO interliggi_k_Page_001thm.jpg
bec85c92d4b08cf6525a7e942afcd07e
3d0cf91f140aa7a1363158b95f35f25f87ce21b2
50632 F20101118_AABCNZ interliggi_k_Page_131.jpg
4392c1e959454a54d9d114f5ac53c1e2
957726d070ef8f55c5b90b5132a4b765d88362d5
F20101118_AABBKX interliggi_k_Page_088.jp2
cfbb183ef340bf1b0c554791345150b4
7b0c65cc559f39d36f80fb792013523f23a351a2
30627 F20101118_AABBMA interliggi_k_Page_144.pro
93834331e2bf5aa7a17b53562df75f2f
93f783a286aa699b5ee2e119cbdf44af652e985c
2772 F20101118_AABBLM interliggi_k_Page_123thm.jpg
3a4e132f94cfe4f65e2b3f1df7f4da17
842b20ed7ccba12e87e42152936cc2f1683978fd
83984 F20101118_AABCPD interliggi_k_Page_065.jpg
6e2b1912ced7882a026ac7a8b95995ee
64ba56c6ef19942f8b94390996a3e0495f0e6ef7
26242 F20101118_AABCOP interliggi_k_Page_066.QC.jpg
c5e65c66a6762ad0ba4fb4043040e963
75043668b68917a3d6a3b542a136bf0c197c6633
F20101118_AABBKY interliggi_k_Page_017.tif
6fbd2f47c1293cb0b7adc65d1ff8c6eb
600284726de2bfbc110a507448e2f63112252796
55033 F20101118_AABBMB interliggi_k_Page_142.pro
a959e0e5f7214a59df3a08369ed9a758
b05f27efd21f433450b3c3ddac504cf949989eb0
26296 F20101118_AABBLN interliggi_k_Page_059.QC.jpg
3b2b5b2cd922d86115e76b8a7b37b59c
e141318f8568c7c4b8d185d1122e8903da72cfa9
81900 F20101118_AABCPE interliggi_k_Page_073.jpg
b0500b6719fa76a7ce03ba773e23097e
8864c57944e30df7ef866737270c79f0147e2ef4
47753 F20101118_AABCOQ interliggi_k_Page_104.jpg
16ec79a8ea02c9722d7bf0063d62c7a5
63b9591fee3523839d3852917e20574353e64a59
F20101118_AABBKZ interliggi_k_Page_005.tif
46738fa21762a68f315f7f5c50167fba
42a88e48f85937d1278b92379fb511bbce65efad
F20101118_AABBMC interliggi_k_Page_142.jp2
a46375c9c4d4a3c3106eb388354377b2
4e89ced634bd5f10d3e4cecf7bae87b88147a040
F20101118_AABBLO interliggi_k_Page_103.tif
d14469b88e37c47fe40c244e62e1ba51
e92494fcb3ed12de65d5c7d227a0db59d3da5413
85771 F20101118_AABCPF interliggi_k_Page_115.jpg
06054b473f593b864d0f6e9d9acbfc00
d53512a9bb56ae7a010bc06aa99061aa854247c5
4439 F20101118_AABCOR interliggi_k_Page_052thm.jpg
9beb0a39e990e76cd0b4e9e7dfd693e4
85218b66fbfc32c2a5acee43cfd887e887d97c73
80923 F20101118_AABBMD interliggi_k_Page_037.jpg
731064f15b5f0cff0407721e3e4dcaac
52d7966d01c6a7bc72d090be10866d8b78eadede
71640 F20101118_AABBLP interliggi_k_Page_008.pro
64e8ee849a668d5edcf7b8faf13b85dc
2ebe9b326ffb155171fe9a285b8ebff31d6429a1
87665 F20101118_AABCPG interliggi_k_Page_117.jpg
f0c064b77d84c32083962f0a5acabbe6
bd43247c583fd6b962e5b3d068b39518f45c0a53
181019 F20101118_AABCOS UFE0021656_00001.mets FULL
5ecec9af120e51ca363c909e2cea92cb
ff0badd43aa46e6e87f9e696619db5af149c124e
24055 F20101118_AABBME interliggi_k_Page_108.pro
397fda3670af0349aabb0f9d022072d2
e2e7d1c5c9541d1e092f941218d98b6be613e9d8
1097 F20101118_AABBLQ interliggi_k_Page_085.txt
8c7fd147e2e63eb180d3365fce0a2154
82c71c3c1d7962e5a7e3fdee418200d6b81168e1
49237 F20101118_AABCPH interliggi_k_Page_144.jpg
75787a259cf0043d27100c42f906efe4
782867a5e6a82a4fa19720241dd33098b5ffe8ae
81576 F20101118_AABBMF interliggi_k_Page_023.jpg
0267664e5916fab20bcd22136d0cc489
a16c693bf7ddaeabf108a5eea7a5d5045bc1c716
2507 F20101118_AABBLR interliggi_k_Page_111thm.jpg
5babc48a1fbd097231f1eef47af3e82e
46ad14a93faa2f019a7163f6b5c6433855738d29
98621 F20101118_AABCPI interliggi_k_Page_153.jpg
db0b893ce4698f4862f936c2f3fe73c6
4b36875e88865d9a8512b3bd9c52f0504155b8bc
392538 F20101118_AABBMG interliggi_k_Page_081.jp2
2ff5f2991e810a32409ff0b87e890e46
c29eafc922846657162db2931920837788fca39d
F20101118_AABBLS interliggi_k_Page_098.jp2
03db93f6e8603005dcf8bb948db8a61c
fde52dd9a10fec54dad1f68ce4a5b5fc652c493f
29130 F20101118_AABCPJ interliggi_k_Page_002.jp2
225a471c49091f910602535300d634ce
0e32fc6669969068a2b7466f946a47af00d848b5
9385 F20101118_AABCOV interliggi_k_Page_003.jpg
d566d0349413beb11d95bb152fe4cdb6
0bfc788a8247ecc40b6dd27070501a4bcbdf78d1
366 F20101118_AABBMH interliggi_k_Page_106.txt
c677b67420eba1e77275b3164c371def
57988b98ad7cd5ae8dbd5edd14303f2390e786ec
1454 F20101118_AABBLT interliggi_k_Page_010.txt
73b5afb2e8f94c4cef2b76331d3e5102
b0bb1f32a9f4ccba5fa473f7029f493857b3292f
763490 F20101118_AABCPK interliggi_k_Page_007.jp2
8139c46b837b7bc52007f56569aab522
57bba82ef76095975bd41ae7108e612994dd101a
80613 F20101118_AABCOW interliggi_k_Page_012.jpg
5b092641a9bcc5648b0c8ab0e134b5e4
b5b3b1b8abd4d6963acbae4993563ef2fa2285f8
83193 F20101118_AABBMI interliggi_k_Page_025.jpg
f85611ed789e0c5cea7717242f2bd5d9
2fb909885c5e4c57055fd9288f1f8f995da29476
480 F20101118_AABBLU interliggi_k_Page_123.txt
b6ba4385e95b1536155d5ebbeab1f5e3
46f235489f334e6165162164fad4634e08d7c53a
1039586 F20101118_AABCPL interliggi_k_Page_019.jp2
8ec021bf01493fda5432003c881da023
a9ea12a8d0cb0dfdbd3304ad37e7d9b29127797d
50902 F20101118_AABCOX interliggi_k_Page_047.jpg
aac1812eed4e33ef088429467e46c94e
12a71fa4f0eec00ba4805e0fa926ea10e88a68f9
15501 F20101118_AABBMJ interliggi_k_Page_056.QC.jpg
d9286f357ea91c0cbbd575fcb603089e
5b82f55956ddb8c2256fd847ccfe414540cdd9db
26740 F20101118_AABBLV interliggi_k_Page_095.QC.jpg
e1d6c03d3787a345b8d0a9effa980c92
eedc239f0928ae7481c9a3ccb5245ff17838de05
F20101118_AABCQA interliggi_k_Page_149.jp2
288dd81cf1ea168a4144712f4b981908
cc312acca7f02fd0d306fa180aebd487df6a0351
978765 F20101118_AABCPM interliggi_k_Page_020.jp2
018e7f658618dcd024fa675d4f366e2e
f15f57048e57387f66a1b396bc1f9efd1c18a219
F20101118_AABBMK interliggi_k_Page_047.tif
f9396ba090a6516468bfd8284894a90f
9a2d7dd9d2b74e1e611647cbc669594ea354b158
F20101118_AABCQB interliggi_k_Page_002.tif
f384bbe85464857b225ca9a5f8257149
f3a55718b5e69ae0512926eb153bdeb761edb77d
F20101118_AABCPN interliggi_k_Page_021.jp2
ef8cc1676d68592c22363b24236dac4e
dc6b5f3c50b775518b9e33e6ea5891514105413f
70699 F20101118_AABCOY interliggi_k_Page_051.jpg
78dbbae709295c059ad7087c70d46d91
b2ff59667e3538235c93c8aa2233d47a448c1d82
1051981 F20101118_AABBML interliggi_k_Page_105.jp2
b35313abc9efd65277316f4f1e2c2fe6
bcc754b9fc34b83bd521c55ba8c7ead2fa294440
27483 F20101118_AABBLW interliggi_k_Page_070.QC.jpg
23bb091d2091c4c44cf27e43273430c5
800cb6c288b822c9bba24c00f57605b092561d8f
F20101118_AABCQC interliggi_k_Page_009.tif
ba51f36b32d8c759e946dbf040f71c1b
7cf604193426e9e4ca7d99f6712e49c72b83448b
F20101118_AABCPO interliggi_k_Page_025.jp2
f78df36fc96c059f868d4fb2a8cfad05
4b56bcd53c7239abde8ed054deb2289466e2f75d
60132 F20101118_AABCOZ interliggi_k_Page_055.jpg
c1b61d832620e27af9d3dd1dc39b1a27
204ccd46f47f74593e0ab55d8bfdbf5b7f29ed15
1997948 F20101118_AABBNA interliggi_k.pdf
2d53f3c6b76aa2f99fd028dd571e3ac5
7cdc16ff4b839dd1553060c21eee5556471b9a22
4165 F20101118_AABBMM interliggi_k_Page_005.txt
aaeda07c353493b1b91ae30033df9282
2527fa067d38b0cc0915a7933c4774b6f9f7c760
28816 F20101118_AABBLX interliggi_k_Page_034.QC.jpg
3ab7fdcb87261514da9deb3774a73d11
ff954a226f516ff5a61769f6b6801d7a385b7470
F20101118_AABCQD interliggi_k_Page_020.tif
4135c01b48120b3c6d9b187a94f82e75
d31f39af4c6c916417ab703bdcdd2502bd081e65
F20101118_AABCPP interliggi_k_Page_029.jp2
b35da37edf2dc503f125916705429c15
7b42aa73a3088d6ad4356e2f60282c47ce522d55
2357 F20101118_AABBNB interliggi_k_Page_153.txt
eedac251c330b1046674152a165eb506
0f38d516ee3a91550a8401873bcc4ffefc62aded
F20101118_AABBMN interliggi_k_Page_141.jp2
a59e192816460d77badf33f584f6f028
670afcf17e0577f36e39b99dcc26bb582bfb035c
6641 F20101118_AABBLY interliggi_k_Page_087thm.jpg
a41f8f26c9acef1bef54003cee869903
71db9da95b81f9bd6ee039f572fd82d7c448813a
F20101118_AABCQE interliggi_k_Page_022.tif
63a862daa83a279b0abe81dad1e66d1e
0d0b856531f81426fe5c5c69f608e103312c16f5
1051899 F20101118_AABCPQ interliggi_k_Page_039.jp2
722349b505f2703cb3ce878ec8375c7e
3faee8f13ceea54120082c328b2cf46cf27f971c
36213 F20101118_AABBNC interliggi_k_Page_058.jpg
13eafbaf901702ac0f3baec41cbacc78
c16e9e1ee66ca0dffcb7150e2d2f43d05862f9f4
2566 F20101118_AABBMO interliggi_k_Page_148.txt
25a218cd4daa239760db640045002400
3eaad76f1bd8aefb658babbc64314f2edbfbb353
F20101118_AABBLZ interliggi_k_Page_151.jp2
e2d4e7e39a37037db131fdcb72fcc8ed
3a33d0ee937804408c1035f17ae6f5d1a7d63d14
F20101118_AABCQF interliggi_k_Page_025.tif
7679428d37628b933652d4fdfd5627b2
fd0ee86ee3bedc985e0b177bb5c9782d82778668
1051955 F20101118_AABCPR interliggi_k_Page_044.jp2
0d6c69a1d2427b94d6749b3cf877e759
b59266c36b0e16000a58da38bd517df91736d667
6794 F20101118_AABBND interliggi_k_Page_033thm.jpg
daf70ab506354cfe1e95297a3b63a799
21f9f3b1aab28978b639d4bda81a75add23b6d0f
53413 F20101118_AABBMP interliggi_k_Page_136.pro
282a0d00a63ef8259dc7ae00e4d4625c
b9fc28b618c6c181d444e5dd1ec319681aa5f7cd
F20101118_AABCQG interliggi_k_Page_044.tif
48e111d1dd7f256aa69adc42bcb5324c
066a3368b733085fc6539eeac2f0bc263a82c647
1051971 F20101118_AABCPS interliggi_k_Page_070.jp2
99ed256f0a054f7adaf127493340d2f1
de73b9b4733e0bbbf53c86d398106dd3867dbd19
27422 F20101118_AABBNE interliggi_k_Page_016.QC.jpg
8da505bfa54f98790ce98f4923601cc4
c275d39dc4eda8940b25b09d695baed95564b94b
2151 F20101118_AABBMQ interliggi_k_Page_134.txt
d7d50928f1d3239d5f4d2d83637ec6cb
4f0d8514be1bca635c9c8a28f90ed92c88c4c47d
F20101118_AABCQH interliggi_k_Page_048.tif
d5c44d1a0c26dd60a3937e0481acb1ac
e886e23d232b367c139829cd73bf054e91f9e311
914986 F20101118_AABCPT interliggi_k_Page_075.jp2
e4b26c2ca8ee54ca73ea81eaedc47a53
a1de7cbab474fb7200744085b30884fe556561d9
5980 F20101118_AABBNF interliggi_k_Page_086thm.jpg
805e5fa6bd31bd0dade472203744bf2f
e264238b818320fe622fea7059f6d673cb93f982
53064 F20101118_AABBMR interliggi_k_Page_017.pro
d4f3fcb70f0b2e6c873fd1a39509a404
375dc0c4dfad6df0c5880c8341859eaf2609a831
F20101118_AABCQI interliggi_k_Page_061.tif
6e776d9c46cf190d1e570798f2e5fa27
1ad68bb59b02f02199118b54bd34eb2680aba69e
623885 F20101118_AABCPU interliggi_k_Page_080.jp2
a6c6691be544d96d141418c4b7a3e8a5
ba59715cd572bc01dcb555224aea9abb3d09ea7d
2195 F20101118_AABBNG interliggi_k_Page_096.txt
cb56901c51933f317d3902b24871892d
a08c7ce8810d1eeeb9d6ad139901c8699ce1ff82
2145 F20101118_AABBMS interliggi_k_Page_015.txt
44e423264e3d56b03e86859d3421cae5
eac9f2fa63db551eb7f6ecf744b6597583231a91
F20101118_AABCQJ interliggi_k_Page_073.tif
165dd0227156b9d3727f6b27aae8b8dd
61398b52337612108166121244cc62c4539eebe9
F20101118_AABCPV interliggi_k_Page_101.jp2
6e836299c37fbcc7e25231e1d40e358a
f350d25d15bbfd0a0068dba0021daf24110c70df
53630 F20101118_AABBNH interliggi_k_Page_015.pro
059727ee6ed0ec36907d303b05ddd91f
5e2b6af3bdc8fa93650fb2223ce01d943b839eb8
3961 F20101118_AABBMT interliggi_k_Page_107thm.jpg
a617991246c30b8957534acf4016e965
0b3d23eb2713f2060aba20d77c290908c1a8d161
F20101118_AABCQK interliggi_k_Page_076.tif
2382622b4452be0091fe7232e7f1155f
2b8c428832e275cae98eea5b65cdad3e99c9c5ff
F20101118_AABCPW interliggi_k_Page_113.jp2
883de1ac89dc64b6801467cbfd2b75e3
243bcbd6e7666d1e75e9391672d4c310ace2f9c4
41929 F20101118_AABBNI interliggi_k_Page_048.jpg
641b439b2cae63a0cb9dc1f3a839fd1f
9449d2ea08ef5542ddee014f18449b5fd431eb89
2138 F20101118_AABBMU interliggi_k_Page_033.txt
f8f67c67e9cac86b7a8403632ad34836
c924362d8480b3c36cbe9bdd9565862933ffcd48
F20101118_AABCQL interliggi_k_Page_080.tif
7f1bff970b63d8b3cea9940bfac718d8
3170f41a885f0444a2f4d2df4e310d48f4764298
F20101118_AABCPX interliggi_k_Page_120.jp2
242ae4cf0a813774fa3b9a53ac43d4b4
c9b3ebf5df304321d533d26001695ed94ed8a846
90586 F20101118_AABBNJ interliggi_k_Page_003.jp2
9d233a2e04b1256ac80ef81c52a4845b
54d4b032cdea28c83e8bf8092f45384424de0bd1
393726 F20101118_AABBMV interliggi_k_Page_123.jp2
f1bba3f41bb559c93b6160555da13f83
d5d04ea8b6df6ceee802c40c930ee28f7386d12e
46658 F20101118_AABCRA interliggi_k_Page_040.pro
460d3b83d3ce6eabbb66f4bceb6f2b1d
fa448a03302d4005b4bd49b7f33bc851d42df994
F20101118_AABCQM interliggi_k_Page_085.tif
4aa48156caad7db76ef79d02af06b860
10c065dc8d766eda66a3d1c659dce7b7f81b00c5
771032 F20101118_AABCPY interliggi_k_Page_124.jp2
fce0c32dcb81a4b24393bcddee5a8e01
a14b1e984ec2abd2cf4c6c7ed2ef20dfd3e1df6d
F20101118_AABBNK interliggi_k_Page_026.txt
756e6e008a48e5c2560bfa20c6d16e2f
044600010ecba4ff2b7f1446bcda7de2f9087d6b
1015 F20101118_AABBMW interliggi_k_Page_013.txt
05309222ee882e7d48cf87d299503fc0
2958d38aef069da103cd3339aff59c58dc0bbdce
53635 F20101118_AABCRB interliggi_k_Page_042.pro
4ced91876d255cfb0bca388c17c336fb
d0c92e090e48d1c72e4c0cac97fa09765a1aa3d9
F20101118_AABCQN interliggi_k_Page_097.tif
0cb6a0b6ff69c448cfea3160bd847729
864dac0aecb5e4f99168160fb8103cae501990f2
F20101118_AABBNL interliggi_k_Page_117.tif
2ff63a86f2578f485922a983b3d8d260
d2618cae44268cc34f6eb25c77942103607d2887
21850 F20101118_AABCRC interliggi_k_Page_049.pro
a8ed53e55028390625971fd734d71ceb
63e031581a3ff69fa013290e0cf90447852f82b9
F20101118_AABCQO interliggi_k_Page_100.tif
c2d655b82d6a051d3af9a27b0b3c3fc6
405ef1b8a7ea60a839f82d7b05177a65c82dc446
479879 F20101118_AABCPZ interliggi_k_Page_130.jp2
91693f0bec414038ac8be19ddc4cd3e2
662c433fdabb5365da1b2eb653db4e59a06ff7d3
28511 F20101118_AABBNM interliggi_k_Page_017.QC.jpg
b0da92a83629adcee6be8002f89f531f
a8a292ecb9292db922b38fd738d37948f27801c5
116488 F20101118_AABBMX interliggi_k_Page_149.jpg
ef7eb5d3eab7bbb7656e5c30592bd473
90778c17273f70fd957f3bcbe7dcfc96e3780bd5
53558 F20101118_AABBOA interliggi_k_Page_071.pro
704cda861d1221b5b980822f23de97d4
781e6cb1c052aac2c4a8f779eba94a483e08da57
9574 F20101118_AABCRD interliggi_k_Page_058.pro
031622d47d9cf0a331d95cf93a6195e8
208b3c5fe35d024e04d7a70a7be8fa154df58e89
F20101118_AABCQP interliggi_k_Page_101.tif
5c54dda834f30e4e3909db3cde24bad4
4a2e3773406b93f3aa0b0e8d2a70d921f17262fc
2831 F20101118_AABBNN interliggi_k_Page_009.txt
2143b9a9ff5573550cb6b08bdc5f0ec4
3d92342c83fc4ea252a165c08712a541610866d8
6382 F20101118_AABBMY interliggi_k_Page_133thm.jpg
ed1fa7b3a964cca860721a124eeee263
6734ace9ce94779a69d35c9e5da937bab43c585f
24264 F20101118_AABBOB interliggi_k_Page_082.pro
1950c01cd707319167713894404bc278
222ac4ab6ea07ae2983a1e14490521299d781c65
10777 F20101118_AABCRE interliggi_k_Page_062.pro
c71adf0471c1211bb0cda30f661d8953
8a32f8e2bc1a5d032c9e6343d527462ba361afc9
F20101118_AABCQQ interliggi_k_Page_138.tif
56b473b83aa31c4c493ca5509a47effd
7c705ba3e1a918e90b4e294f283923dcdf044e01
103 F20101118_AABBNO interliggi_k_Page_053.txt
d344933930530753143b1e865b86bd86
5c704a74188f897ef4fd6d5df67bbb69a9353709
87857 F20101118_AABBMZ interliggi_k_Page_043.jpg
fb12b5bbfd59829fb58300f22ac1848a
d198631c4e83ce558c5e835da5d9752da9ec9f3c
54666 F20101118_AABBOC interliggi_k_Page_027.pro
91a30dd4e1ee2ef302dbbef3f6afe0ec
bae1c94dea4087ebf6b1288e6107791cdcb5d575
45593 F20101118_AABCRF interliggi_k_Page_063.pro
a60ee555ac5fb401a547de42baf62244
9a5ce24e2a9754cc7cd1736276a6075158ce2f4d
F20101118_AABCQR interliggi_k_Page_141.tif
790ee48e8f3faca1981e94f63263f791
a90da764b0700125c49c7f93bbfe7da1bcdb5069
6759 F20101118_AABBNP interliggi_k_Page_092thm.jpg
4358e4da5ec0a9207bdb6a3ba0791f23
f8952ae2eddd6f8482cb2e23ffee9eec41230af1
F20101118_AABBOD interliggi_k_Page_127.tif
d6c596ca2ca18474651e167f1045360a
23e97621f057a8c5cde19f0c8c0a34f39a5fcc1e
53039 F20101118_AABCRG interliggi_k_Page_068.pro
fef4e860fa909a376bda36be771b4317
c35a1f69a834771acfcc3a470c696c5995765b7f
F20101118_AABCQS interliggi_k_Page_143.tif
0ec3a3d402e3dd787f0bb859bdc058eb
b94e8aeffec6de3d2e09dc49779eeef78ebbe406
1051954 F20101118_AABBNQ interliggi_k_Page_152.jp2
70f5d5f87fed12e8e4b837b8668bc469
f723fe11e67a8c2d9ec12249bff14122a964e528
36596 F20101118_AABBOE interliggi_k_Page_078.jpg
0e8aad9663178e206f086cca08efa0bd
078f526c18ff574aa32ce323fbd0ab1eac3ab18a
21021 F20101118_AABCRH interliggi_k_Page_083.pro
cb7e21232614bc00d0c173d70440093c
e47ce77ebff03d1ee44a76ebcecc47d5d70883e7
F20101118_AABCQT interliggi_k_Page_144.tif
f790a56f7c85766a9252ee6a6eaab872
e77182c1b8af802069764fb0d0e35ac6daacf69a
3717 F20101118_AABBNR interliggi_k_Page_060thm.jpg
40433c8a6dab0af400b3aef74a557f0d
62ff415e9eb4cc84f1a00c899103275c635630d8
F20101118_AABBOF interliggi_k_Page_016.tif
be9c0e9157bd5952d9b6b3ca6919f826
fdc3934868b3fb3b6f287e8ea1b29dba50f3572f
49140 F20101118_AABCRI interliggi_k_Page_088.pro
14616db3667b38fae3682348dcd25654
2a54872f8e26680187278007891c86f84e160d8b
F20101118_AABCQU interliggi_k_Page_154.tif
b6e25806239a94764a9e8e0d4327b744
f54f913f2e7a62b6d781ff6b22de1bf6fb61bc71
14536 F20101118_AABBNS interliggi_k_Page_128.QC.jpg
c3435d1b15815105ddcc53cdca9f5248
76a969776b562dff953f76135d48db8bbf8a3b24
553 F20101118_AABBOG interliggi_k_Page_058.txt
cb744b94748444367d543bf973b90630
92908be5e2d5220dd3d95fe25df05d512e15bec9
53242 F20101118_AABCRJ interliggi_k_Page_090.pro
92383721b4e0da9e3771bc8e7392a3c3
658e323889262bde0588ffa26cde9443fb637338
1047 F20101118_AABCQV interliggi_k_Page_002.pro
da6b67188df7a9002216292753f3896a
0f52b6864f9645ca374f30b1cca102056466c4e8
56968 F20101118_AABBNT interliggi_k_Page_075.jpg
00a73488ebb31dd8e9ab14b8f1ca29b8
53ebe721d14d6f563512180c74bbfd239c375b9d
F20101118_AABBOH interliggi_k_Page_118.QC.jpg
fef7a50474310bce8abace64af551805
158d0b1222d8c10a20022ecd2e324645a78295fa
56228 F20101118_AABCRK interliggi_k_Page_093.pro
a448cc9dc9c0100cfb4e2f37c2bdd1ba
92d7096bba25117d685e997555e29908909c8d71
106401 F20101118_AABCQW interliggi_k_Page_006.pro
2371472959a9ed101e4089892d61376a
34f9680b1857d3bf449cfd1aa9e39801ae473f51
51637 F20101118_AABBNU interliggi_k_Page_025.pro
f37cceb4cd8c52d0586f668a2857767f
1c698bbc84a62c61e2493a19b533cc20cb6ce4bf
F20101118_AABBOI interliggi_k_Page_006.jp2
3a8b14c8406f7813143a868bc11b3378
de5886b59d832e53ec10be3665db6f4e0892f12b
42147 F20101118_AABCRL interliggi_k_Page_099.pro
34dbf4598261d426e28671a97c6ffbe9
575b212061779afc8edf9150ade7d3597c8163ae
19304 F20101118_AABCQX interliggi_k_Page_011.pro
f9f08f0ebeedff383950e15d1f9620fb
ae92257486884beb36cabf4bac498ae523c904c8
4575 F20101118_AABBNV interliggi_k_Page_074thm.jpg
88cb89bb1dace774712fff9715fa354f
07d014272ff961c468c39a5f120a8112e5c5cf4c
1051926 F20101118_AABBOJ interliggi_k_Page_092.jp2
c0f3a0cbbabe75d972635178195ba67a
c6793f44c368a2b7a116506288b92593997dde62
478 F20101118_AABCSA interliggi_k_Page_132.txt
dc99be09c007afc9b9e62686bb0c4645
8b804fee736e69ae83224cfee2f4ab7578cc11bb
50498 F20101118_AABCRM interliggi_k_Page_114.pro
e748e95630c01bcd0c78591aef868e16
5fa098550edd1d9bf03fee91bed2085804b5c735
44569 F20101118_AABCQY interliggi_k_Page_020.pro
b4c06c06f9baceb6930c175702a4c911
5e90368013d1ab7f49c312a18d546cd14dc30ece
90470 F20101118_AABBNW interliggi_k_Page_018.jpg
18a6d2dcf402e277cb381a29af42cc0f
8e4c39e1d9205cd003ba71bbfd1decebfe41fd05
2175 F20101118_AABBOK interliggi_k_Page_025.txt
682b68ca3ca9e67e1c320e5db9dcb1be
daf77ddb10bbd13cb30a3c5a357770fe451c280e
2317 F20101118_AABCSB interliggi_k_Page_141.txt
f604c6ffa38a1045165c727dbed8581b
a7cf1cb0b260f6b1df33f1469bdc9f6f382bd7ff
52752 F20101118_AABCRN interliggi_k_Page_115.pro
8cb419e728ec67ae4a4bd33b742f6364
b66e74cd4567abd2452bca64c41a9a3b0f804da7
49021 F20101118_AABCQZ interliggi_k_Page_023.pro
dcde8be4ee5d533167d4e21f05d8acfb
911f1157fd321097caa8cedfca54a9a4126e0485
28281 F20101118_AABBNX interliggi_k_Page_136.QC.jpg
cc965533cefc2685c9d42da8b3cc5185
711fc6b60330bc846e3ae51d4d4df7f4478d7592
2062 F20101118_AABBOL interliggi_k_Page_016.txt
fcd0fadee802c598bda6576e96cfdb48
27d7c9c60cbc9c30df52d881f9d640b7251afad8
234657 F20101118_AABCSC UFE0021656_00001.xml
7ebde49acdb2de293cabf7c03fa2b760
9ca6ecc48d46faa0bc8f7ab59b92130f6e35548e
52387 F20101118_AABCRO interliggi_k_Page_118.pro
892b464dce603e63705d0d0cb1edd025
5e280fc571a44cf569e896ae8eaf8cb4d48b332d
52427 F20101118_AABBPA interliggi_k_Page_087.pro
5b31fd24be2bad2a4306a18b8b239603
670547f3506eb3aa9e807a09bafce4d45cbc9654
27982 F20101118_AABBOM interliggi_k_Page_042.QC.jpg
2b88e7d729842137fc84fd8399f206b2
c5281da096a6cb0f279be8283b293e4712c627a1
6494 F20101118_AABCSD interliggi_k_Page_017thm.jpg
144553782286af95a40c3bf120d74261
8651e6e84be54537da78400cdc24929620067262
46741 F20101118_AABCRP interliggi_k_Page_143.pro
751ec9e2ce9609cde66ddd5f5484ab31
7c54c8805193caa785e241d37d7c8fbbdc1accd1
46117 F20101118_AABBNY interliggi_k_Page_012.pro
2c1184f11ce0eab16d87663649471e37
975653095f704a964c5035faff9447268a6e7dc9
5420 F20101118_AABBPB interliggi_k_Page_076.pro
02bae0c1b4e8495369f69ecf391ddc2c
5e6c1e6e2dc49bdfb7f76af2b518dd7e96f07d07
1051906 F20101118_AABBON interliggi_k_Page_118.jp2
f9a4f7e91885f541f5ae8e1df77e8913
6fc2eafa121a5a8ef2729d4a711d0019620134ad
6593 F20101118_AABCSE interliggi_k_Page_024thm.jpg
bc66ea9b0889265bc6c165e261876668
d0ce796943a6522f73810d89878794ad57de3db7
67772 F20101118_AABCRQ interliggi_k_Page_150.pro
4439e24b76feeaf93f3031087526c889
2f0668539bb032eeff6287819efdaafea778d00a
86447 F20101118_AABBNZ interliggi_k_Page_068.jpg
35f2e8a96f098c8ec64f708afb473065
4640edda77f8014957eb94c3f1d7bc7b4e38303c
23679 F20101118_AABBPC interliggi_k_Page_109.pro
98006955e0076e7d037bf5909d7a0c0b
cb452fd50f80d29b5f180eedd2a5159d2e6cbf06
106664 F20101118_AABBOO interliggi_k_Page_009.jpg
088939a7212085b6b13d62a6899825fe
38a73aa110f0a36903459547b7eb19405f04f19a
27150 F20101118_AABCSF interliggi_k_Page_032.QC.jpg
a4e2cb27eb978b9b00373300029ecbff
9b8e1ebc47ebe0eabf4669bd7f16167de750894e
2061 F20101118_AABCRR interliggi_k_Page_036.txt
79da87502a7c12593e47076013bf89f3
4e3441599a7be0bfef56a95a6b3cc632b6301689
602722 F20101118_AABBPD interliggi_k_Page_048.jp2
fa7da2e4e4062c6f6aa77bfe751af5fa
ba691b4d7269b5b14944ed041f10bfbe16666caf
21230 F20101118_AABBOP interliggi_k_Page_049.QC.jpg
99bef3377e9630c841308149bfad6150
c7de7e660a5f8dfb09043a7db059499753b3f523
5868 F20101118_AABCSG interliggi_k_Page_049thm.jpg
a30af1fe5ad5569efea6167aab9f3176
2ed3cbb14850c503ffbd23af7c4a9c33debc2205
557 F20101118_AABCRS interliggi_k_Page_048.txt
70afcc34f6e78ca61995c01ccfb12419
e79e9635c7b96ffe010e4971fcf224329f7248f6
84372 F20101118_AABBPE interliggi_k_Page_066.jpg
28aa7e1cbcd443100bc4a4775254b3d8
d4492c279eac2be9d3c0bbd540d10b4610b80e28
F20101118_AABBOQ interliggi_k_Page_012.jp2
b9cc19fee34e9b16e15d20185ebc5af6
88ff4ab3dedb25f5b76ac48af807057d8cb60d3f
5485 F20101118_AABCSH interliggi_k_Page_053.QC.jpg
57459f5855a9f324c7f267f380b7ddd8
87d6094789e184a42c4940787bda1c9b0f384df0
989 F20101118_AABCRT interliggi_k_Page_054.txt
b5017dd50c17d23a8a7836682df7cde0
12002d4db59e8273c5ab60f20f2652d377306b10
29041 F20101118_AABBPF interliggi_k_Page_097.QC.jpg
7cc0679e5b0c3efa21ad8a8856a4adaf
279cbdfd5479c16acc4d532c92b486a2e52ca75f
F20101118_AABBOR interliggi_k_Page_074.tif
29802e145a072b8ae9d6613fc92cf665
24b4a2ce3a175dc71c42014e3159db2a5e2728bf
11686 F20101118_AABCSI interliggi_k_Page_077.QC.jpg
21b72f07ff3940bfc2abe89df4f87aac
39db995ee4ccf74ada45bd475fb7db3ca821e1e1
871 F20101118_AABCRU interliggi_k_Page_055.txt
06e269fcec370d0587e30bfe5981a735
9e2324a1b29ab598bd2ebc4fc4dd82f0b9834415
1051946 F20101118_AABBPG interliggi_k_Page_090.jp2
b41f27f8ea6e8ab55ee24891068549cb
ca48fde2e778dc2cca017a95c83a432096c6b3d5
31661 F20101118_AABBOS interliggi_k_Page_046.pro
af6361cfc72cca39c5595799d2a7a2f7
0377995eed81e21e61a7f81eb78b9ecf36e9ec05
16906 F20101118_AABCSJ interliggi_k_Page_085.QC.jpg
e3bd365683ce57c1cd4c58365111a653
fd04ecd7b3c63e5859ae2293a0e61a3a94325631
2017 F20101118_AABCRV interliggi_k_Page_064.txt
cdd697a4a4da8f48e42c3a83e9f94da0
3c6348ad0fba2191c5fda83a0ee1254756fc8893
65197 F20101118_AABBPH interliggi_k_Page_152.pro
003eaa116b10f8d6c5789a811e741960
b327453024f9d0d5bc4da7329b40b8bdda5b5c35
765 F20101118_AABBOT interliggi_k_Page_103.txt
2504ab49f3917f69b33bbf9b75cfdac8
7fdec8ffe4e0d132f497d95a9661d8eceb1eac2e
28352 F20101118_AABCSK interliggi_k_Page_096.QC.jpg
4ccce9dce39c9eda602db9690a98c5db
974f469fc4cd82d27b426a3a2b5138a9fb15c9a8
726 F20101118_AABCRW interliggi_k_Page_081.txt
da20da6488a24ecc214fbbd3c1679d51
2759965467605dd45c4ad289739db787bdb03456
49563 F20101118_AABBPI interliggi_k_Page_032.pro
7e3bb5e398ac1ae18b528ec5819581ef
a153fed687dc2e26ccdfb9c58e29e6509d4ae25e
55676 F20101118_AABBOU interliggi_k_Page_045.pro
ae03c6658f8259d271892f912afcb5a3
a0237975fc97b2d4a96a6fe24604bb20821ae5c6
3064 F20101118_AABCSL interliggi_k_Page_103thm.jpg
ee37727330f2bd8c406cb29d18b62082
e7a4d74b7f15360ab064611cd8c7f780643bb93f
2088 F20101118_AABCRX interliggi_k_Page_090.txt
0002b01703daa771bb083ced5d891f03
1331958fe9704b51cc50f86b0772798d8702fc65
555151 F20101118_AABBPJ interliggi_k_Page_058.jp2
0bed7d2dd80654c5579ed3615b6afba5
56577546734a289b2098b3d0bde8fb86adaac136
6521 F20101118_AABBOV interliggi_k_Page_121thm.jpg
c9b9354b5ea942bf300fe33522b299f0
a16e0601c3ca2047d9edba5c2bc8af3c236d163e
1905 F20101118_AABCTA interliggi_k_Page_154thm.jpg
543d0ea8bf500c897f6a4958b7a39723
ede4305f5512a5878dc40e1722b430ec756264ee
10653 F20101118_AABCSM interliggi_k_Page_103.QC.jpg
d014758f1c7603dacaf3ae2aaf6f248b
7e117dc18fdf93cedabb2037b156c787db30a7e7
2222 F20101118_AABCRY interliggi_k_Page_091.txt
fbe59a29c8c06b78829537df46c24bf7
d024495953f5edd7b27812c9f36a91faad91bc46
85990 F20101118_AABBPK interliggi_k_Page_029.jpg
192e47b14d728cc7d4d2ce0d7393893b
739692b6eeacc41283e98c2e04b1c8faee627209
600 F20101118_AABBOW interliggi_k_Page_126.txt
a842f386355a73fb68466de269d57445
ce63f7bb38281ec2e4e223ab5685163ef10252b4
13577 F20101118_AABCSN interliggi_k_Page_104.QC.jpg
6a0046a70cf1db87bf9ef4e0f88f8731
3589de8bd6ae0e5227322b425a835492b5544dc5
2118 F20101118_AABCRZ interliggi_k_Page_121.txt
30509268b3d76f1134d53e33c5ee88c5
7f5629f52cf264dcc3319531e961356002f066c8
F20101118_AABBPL interliggi_k_Page_089.QC.jpg
d05745c91e416043de101c9992f172eb
af3188f6042ad20c2376720ad9783bb8977b1bbe
F20101118_AABBOX interliggi_k_Page_123.tif
2a76a7c28f8f750e13879b0db393f143
b26fa8c1b324396a025a801ad7418e28c9994999
5218 F20101118_AABCSO interliggi_k_Page_105thm.jpg
5b73242700356b3df8be21b05ec772b0
2f9a312a977152d1fdb30e2c22b8751f753ca9ab
111845 F20101118_AABBQA interliggi_k_Page_150.jpg
5c422e41b994b23d12c823bf36a38ef6
47336aad52eba45d3f83e747a0316b4cbfb583b6
6456 F20101118_AABBPM interliggi_k_Page_071thm.jpg
fbe9a9d89044be1d23df373b171c790f
429765ac0d98d1c6818e76d0578f05e94e5deeb8
70003 F20101118_AABBOY interliggi_k_Page_116.jpg
a3a97f0353e856460ff2f0d1c3b43a5e
04b007895988f0ddc1084a0301e3ec08668244d4
5505 F20101118_AABCSP interliggi_k_Page_116thm.jpg
80377078804d32f965a53601bc5895be
02506c9f5ecc18e3cb95f74cca75a1e8c4880595
1261 F20101118_AABBQB interliggi_k_Page_046.txt
2e0d813938d2055d6ad59e2ef2146306
363d60e7badb8e563071a73369c5e1ec81b9c498
F20101118_AABBPN interliggi_k_Page_040.tif
1cb85e26e3664e20e7004497ac773cd6
a34278c28208c1df53a1997195483a9e137b7c97
3580 F20101118_AABCSQ interliggi_k_Page_122thm.jpg
ab843615e86b0777362437995acac210
406f876a27697753cfb3bff525d6401d4854ba21
87200 F20101118_AABBQC interliggi_k_Page_017.jpg
bdb8c6172802808697a37da3084c9688
cc99e2fa2e9c7cc6f96f9e2566c719b5a9930d08
F20101118_AABBPO interliggi_k_Page_039.tif
347be8ea0294973bd1339a214bcd5b11
8adf39c18831f3ecb1b4b1dba3e6b732f08e922e
2159 F20101118_AABBOZ interliggi_k_Page_043.txt
1eeb721638c4f8d1ee86b1d423c73fa9
aa070a983dda5e12b05635579c26d4515a36de9b
2309 F20101118_AABCSR interliggi_k_Page_129thm.jpg
d39c0edfd275adbb85da0eacb958a9a8
ca98b914cc6e8add62281a1ccec5c5d86b457142
1765 F20101118_AABBQD interliggi_k_Page_052.txt
195f8b596fc4c6db79bdef2269ee5c9e
3e323510046486ad05ffc5f1638f0c935e2302fa
70443 F20101118_AABBPP interliggi_k_Page_143.jpg
bc35af885abae54fedfe5c6ed0cc437d
93d0c74ccc2392756b56435410144848a31d392f
4004 F20101118_AABCSS interliggi_k_Page_131thm.jpg
d129a22989707fa94335b0fd128c7138
84571ed00fcda31cc68fda49eb0f70a0e1b5efab
26836 F20101118_AABBQE interliggi_k_Page_051.pro
a5584d15731e34aedb2593e0eb7fc62b
1b1f62f3dd07d306a26f143456ae6b3a047ee059
32110 F20101118_AABBPQ interliggi_k_Page_050.pro
2b5f1934fd4d10565bb541f6a4130695
48d3566b9c22158d96533ccdf7d9414f2776a050
14266 F20101118_AABCST interliggi_k_Page_131.QC.jpg
2e7a38a5eb89fd2b3572921fc9102976
d4f4c28b47d30d793239ec5aecef418ca7f53077
2790 F20101118_AABBQF interliggi_k_Page_149.txt
8a66029acd9f8511860644cbe6be5961
d6831fe198c5d6cd9d46a2d18ef1deb4bad4965c
1990 F20101118_AABBPR interliggi_k_Page_035.txt
4eb7ccef8adb5b1ecbdcd233f6bde58d
8c182ee01187f0aaaf2ae594ff5ed27db2253227
12141 F20101118_AABCSU interliggi_k_Page_132.QC.jpg
fd4a6b65f999e4920f444c5a3659272f
4e51e4d32e20e280c535bec2333c377ef3612d2a
49807 F20101118_AABBPS interliggi_k_Page_073.pro
98b9f43bf3658a065fd6d1722c5dedb0
363fe57a5a663faba0fb55edf15ffdafb22eaf20
5848 F20101118_AABBQG interliggi_k_Page_063thm.jpg
a1aef6b875f0c2820b483d215410db12
f28704bd53c2c770895eef336d65b03a88364e4e
6591 F20101118_AABCSV interliggi_k_Page_135thm.jpg
e91c02269f082d432402ea5e21fa6dfd
c92617fead5b93c02bd1bb35245e10d4d8d60103
26139 F20101118_AABBPT interliggi_k_Page_069.QC.jpg
ecff84294bbb2981e23c45253e020d45
ca5fbf4ee2557d48fd4ca14d78778082a8b00107
2057 F20101118_AABBQH interliggi_k_Page_032.txt
588abc3e490ae20e6de2d88f70fb8289
41f88dc8554c521428131784484514f8b3c008d6
4182 F20101118_AABCSW interliggi_k_Page_141thm.jpg
cddf3108bb7ef1da40f731931468fa72
9bdc300b89c59193117a8c3a1bbbe3588749f6eb
22040 F20101118_AABBPU interliggi_k_Page_116.QC.jpg
6e708f9e7b5545a3debebf48fecc3872
aabc60ded4eef58e2dd6ae754bb8ba967b4a86e6
85375 F20101118_AABBQI interliggi_k_Page_119.jpg
fe3670040a0ff1c803fd3ea73547568c
9e0ec5496dfef25b950fde9735078f49b094fc25
19636 F20101118_AABCSX interliggi_k_Page_142.QC.jpg
43213f3782d0c9d14aa05bfc09fc7861
5f04ced038892fded2d264d38a00c7098908cdae
6460 F20101118_AABBPV interliggi_k_Page_021thm.jpg
2cd2ae0059a74b384f10a7e53b1be600
762c60002976581bed3f82dd74ceee7f0d78c121
2126 F20101118_AABBQJ interliggi_k_Page_014.txt
3f78f9fceb2a8bd00afe9432428aa3c7
514e186e041c07ebfd9fa61102c5d74e9da451c0
30276 F20101118_AABCSY interliggi_k_Page_152.QC.jpg
46aecd2e247abc4fa8b1f2d1c87aae51
09de8babd67594ddbe874f9b3c263b47f28289d7
88820 F20101118_AABBPW interliggi_k_Page_070.jpg
3e2a3ca52b1551393524fbd784a17b08
b648d3e26d3800648c21dc4a939f05df7249b930
89177 F20101118_AABBQK interliggi_k_Page_089.jpg
48e9ff03acd0634b3675918cd96fe4a6
3ed4de86f8ea502f74e5106f6b7a59abba665e23
26662 F20101118_AABCSZ interliggi_k_Page_153.QC.jpg
198980fcc9af98306aa34065c13dbd91
fc7b7703869604c259711d9574be6364f503d746
2052 F20101118_AABBPX interliggi_k_Page_029.txt
918ade56e823e59dcd7fa477e7bb3fef
c2a7efc91f489db4545dc2f5ea3960636a060d7d
1550 F20101118_AABBQL interliggi_k_Page_101.txt
1202347db124720dc514c946d1bc1198
5f2d8c023d5acb833fe1492f86146a62475ea2d8
7032 F20101118_AABBPY interliggi_k_Page_152thm.jpg
18d0fd97f502f6fe858da7d6c4bc3cb9
8f94cd5e0bb68f8f72aad423e3ae9aa1f178d939
F20101118_AABBRA interliggi_k_Page_072.tif
6b74f97c08c107fb1dada9e028530fae
c6174c326494238cfd8ac89e9091b53ed14dc5d2
26457 F20101118_AABBQM interliggi_k_Page_122.pro
b6c561f48dd21c3f4094edd4567c017f
cccd8c9e9e9980be07b1d71c2fdb7d6f933126a3
3052 F20101118_AABBPZ interliggi_k_Page_062thm.jpg
21799662eb4b9895f4ef019a1cb9f3f3
4c67e687eabff0beca05bef0a6eb5614199b0c39
54741 F20101118_AABBRB interliggi_k_Page_096.pro
31c898af784cb6413cd7abfb40568e08
e7bbd3394d5e8f9cf15a5b884891939281dab82f
28309 F20101118_AABBQN interliggi_k_Page_145.QC.jpg
cbd436468271ae6f601cb0cb9fca9c80
fc8151332c051281c020cfdbbc218ae5dfe331a8
49684 F20101118_AABBRC interliggi_k_Page_109.jpg
16d1591b4956ef2434284eabfe612c04
b1d84d0ab7b257e769c81e6a8c15359400d54a94
2629 F20101118_AABBQO interliggi_k_Page_152.txt
cfb78362ee48fc52d0e1a66f4f885e12
7fabef77738b9efb125f53b668fa850e91c942e0
F20101118_AABBRD interliggi_k_Page_119.jp2
0712160347e9acea8c53d74d193d3252
bcd28379824cb967fc5aca99ea0dcc08d42075da
36168 F20101118_AABBQP interliggi_k_Page_057.pro
5950dccfae80f3b1cfdf61f25a5ffd29
10385a4742205f2092d1bb5d468659e25b06978b
F20101118_AABBRE interliggi_k_Page_055.tif
c56ce61df164f71991e1aec071e9003e
f0dd161a3b5513f5f6f8d15baaa1ba98bec710de
F20101118_AABBQQ interliggi_k_Page_066.tif
17eed542a510c9df178fd73d523909e1
030e83124b6f9263fe23321f368e2000c98136fe
23699 F20101118_AABBRF interliggi_k_Page_101.QC.jpg
afc1d4f44e9dce553dc024bfd4ab12aa
5051f82bef40edaf46df0ab80c7b4a3204efa967
17114 F20101118_AABBQR interliggi_k_Page_052.QC.jpg
0c393e3f701fce5b271e68ad9b06cc45
7e12867ff136214f4532231100fc2f20f9e7aa1a
28552 F20101118_AABBRG interliggi_k_Page_033.QC.jpg
266eef06100643e1566ced1bbe3ec01c
1eeac8c47f5e2bfaac1e21a52258846a249e4edc
2066 F20101118_AABBQS interliggi_k_Page_113.txt
f2a0fed389f94ce4f107b491f5152239
8ae11ca69da8425717aae2c9d452745269e339c5
3071 F20101118_AABBRH interliggi_k_Page_144thm.jpg
519663119b6c27982eb3c22d66d4e504
6090bf3dccdeef6b36bcf54d5dc6ad209e8c8f19
8990 F20101118_AABBQT interliggi_k_Page_123.QC.jpg
0ff8a5ad6f49ecf5b3184810e91b12d2
83c9d0adf200d8f5ecfb978e6ebb1c42504b2b17
3880 F20101118_AABBRI interliggi_k_Page_055thm.jpg
fbcb1f7e39364baa4a88eb25c2b36bcf
2a7c779a9ea217cb67352437690d623c4eb3f259
1964 F20101118_AABBQU interliggi_k_Page_086.txt
b4df49948f049c4e6f9a23153785904f
703deacb7b786c37a9a18dda5ce7ad488beb662b
54238 F20101118_AABBRJ interliggi_k_Page_033.pro
c08f7ae4f64ed6320c8bdf6526dfd924
4dcc2ca540b84f281c165464425ac94bf6491765
2190 F20101118_AABBQV interliggi_k_Page_045.txt
a8c636994a81614a473284d1fda2684d
8d23d27145ee374eb4f43ac3d8d620df70b48cdf
5385 F20101118_AABBRK interliggi_k_Page_099thm.jpg
55eb70d563b981ba0e1e39ccdbc4728f
b332204ac25036ff5fb2c42e14909d63ecb02bc2
F20101118_AABBQW interliggi_k_Page_093.jpg
f55391618832b40e409421971dea2cf6
29066264d440160ad3ac6927651899300c969b68
799 F20101118_AABBRL interliggi_k_Page_139thm.jpg
2c35591e622abbc5adcc2a9466ba178d
314710bf7f888beea909ba115139eb589209c0a8
F20101118_AABBQX interliggi_k_Page_087.jp2
dd7afaa1f69f862cce34c2805b9d818d
fbc26359d5d7598ccec47a3d21f15ea215c3c798
46113 F20101118_AABBSA interliggi_k_Page_080.jpg
a1740781db0136d7e7375d088d0cbe28
e78f9aca519b372ce25e31ea5868e4b119cdb993
F20101118_AABBRM interliggi_k_Page_114.tif
6a99ad2281096f56f2bbab011f6fa692
3a23bf5d2c941df116ce99cabc6862c06f431d58
15994 F20101118_AABBQY interliggi_k_Page_055.pro
5388e9b09356802af756fa6bf5303146
c845e9f6a5f414a3139b5109c1c1f77942b75832
F20101118_AABBSB interliggi_k_Page_095.txt
0b589b617e1b752d5fc888843b143ffa
523393abb80c6a1eb79c4ffd8a9cf56f3eadb742
722557 F20101118_AABBRN interliggi_k_Page_047.jp2
93db3c0cff7e8a1bd1c73d6bcbd5f88d
1d4b5b816dda0d2a768b8ca93e6ad744e2144485
54850 F20101118_AABBQZ interliggi_k_Page_127.jpg
07a1d683f5f4fb1365bb80f56e0015ac
4806b9d0844fc1fa9f0602fd8382301cb60a93fc
F20101118_AABBSC interliggi_k_Page_060.tif
2d570c108d03392f690228cad2f6e824
7164bb25d8a7fefdf98425f882a62f50945ddce5
6712 F20101118_AABBRO interliggi_k_Page_068thm.jpg
cc359f0214f5c081d0d3eefcf90fdb3a
dab3886babdc7ff3623e02b23f32c840e98c14c2
7628 F20101118_AABBSD interliggi_k_Page_076.QC.jpg
4347307a2001d894ffdf96b87c790c7c
0f6a46d14cc6c92c2b94b44266d56e7e616e59e9
56293 F20101118_AABBRP interliggi_k_Page_085.jpg
9fb5ca65a09fa6439c8ca1754c8eb19a
d40cb6d143fb2b5aa8fa1000ddd01085c0ba340b
F20101118_AABBSE interliggi_k_Page_115.QC.jpg
75ee8deafbfd6276c547c44b52a6c721
2f3445002e463f14c6991340ac86ae1e04f8d58f
29806 F20101118_AABBRQ interliggi_k_Page_146.QC.jpg
2df9c39324d4aa8afb044c453b410911
5b508e4f1551d085550a6f84f4c4edc9d6e4d0cb
F20101118_AABBSF interliggi_k_Page_004.tif
63515c06dc6bcdde6db09517a28d5a15
72a7c3d8ff384871fa32ef1ad0b51ae9b84a7baa
1033262 F20101118_AABBRR interliggi_k_Page_040.jp2
4440b6030d0ebdbd149e65d2f45345a0
990262507a52fc14ddeed647fc84b9beb7d2454d
19503 F20101118_AABBSG interliggi_k_Page_080.pro
d3950ac300fc15e9d4cb0024a77db978
2451fff2405000d0283465786939985d3f3522e3
52067 F20101118_AABBRS interliggi_k_Page_029.pro
fbb4d0b2fe017a3ed9b2c36d984206c4
bd094c390af325a9c4b79e34e7aa33cfe83c5dd8
F20101118_AABBSH interliggi_k_Page_086.tif
7357c536a2d6acc3b1e4bbca7f5ee719
13e6b003140b86515cdae2386a6844575f8cb284
933903 F20101118_AABBRT interliggi_k_Page_085.jp2
e0e3039d5706ff2bebd703ea110bf296
a52dd2aa7dd98db52f0db39985e69c9faf7f2244
3846 F20101118_AABBSI interliggi_k_Page_124thm.jpg
08f6ed336d3c55b5bafce8f2deb07fbe
c34187efb914b93d7d65bee98a4296b749dffebb
6771 F20101118_AABBRU interliggi_k_Page_098thm.jpg
ac51c6a0ce9d96cab37023abc96043ad
89873ed6d9dfaf89914324758625b454266f7469
12972 F20101118_AABBSJ interliggi_k_Page_048.QC.jpg
6dfabf6e2ab5c929fcd467bdfc82de29
16270b1a9035600565333bcce12e6d718b717f98
F20101118_AABBRV interliggi_k_Page_056.tif
2526df5c4e78b6920215eb3985d3f8b9
8a524c6ef70656ac68f089cb53e29e64d3bbad51
52060 F20101118_AABBSK interliggi_k_Page_014.pro
ffe35ad0346fbe41b4a056b746ebfd56
c2af1e1685f065f418d2292fcc05e7fe0a720cd1
50120 F20101118_AABBRW interliggi_k_Page_113.pro
74186af482b564ac6e3877445fda32bb
fb2f21aa0b765097dc1a1d2b54d06ca6c1a18331
88291 F20101118_AABBSL interliggi_k_Page_096.jpg
29c47e31a33aae88a43e043171194c3d
25bc08ee2aab24b9598d2ea35284ddb59d76da8e
F20101118_AABBRX interliggi_k_Page_018.tif
c772dcfe82f8a18bfd2fff2a4b1686c6
b81ffd9375f089aaf6056b46506bbdf0de2dadcb
6879 F20101118_AABBSM interliggi_k_Page_146thm.jpg
c10290841eb71dda7e93d1d5e227e277
0d86030b9c0bea0133715867c2071dca68f0b48f
7798 F20101118_AABBRY interliggi_k_Page_106.pro
dc7eba1f18967c3017b2e3de9bf60627
7d4713375a509c76f66f076c1883223103126a64
3140 F20101118_AABBTA interliggi_k_Page_031thm.jpg
862d66ee2a0e8387d4505b5d69932b5c
7698d3316ac9424103bf45c216bde0e0a2b8807a
F20101118_AABBSN interliggi_k_Page_137.tif
d5ba56e37b4566f54be68bf306696f38
2c13e6546ed0ccb1a8237a03c03f6d812ee67dce
10795 F20101118_AABBRZ interliggi_k_Page_078.QC.jpg
142eff310af50b4fa48e01dcbffbb166
a11308e6e656bb1f8f901e808e1d326caca4dc1b
53913 F20101118_AABBTB interliggi_k_Page_043.pro
2bde0654a7b05757e3953cac479fd18f
130beb734d98ad9c30e5008f0cb7ae039a29d777
53965 F20101118_AABBSO interliggi_k_Page_070.pro
dba741dba19adb76d3a4474ad49c61a1
ec3780f484723c1d62f3cc8b5cd99f6aa5fecf24
55369 F20101118_AABBTC interliggi_k_Page_044.pro
5005afb4a95e312588d1d12d89d514a1
db1a5ad02542edd4cda9eae50eb5002293449251
949003 F20101118_AABBSP interliggi_k_Page_083.jp2
b601164ed3aa16faa19ccc2e5bec5878
6a8023b8808fa0b862b5e23cf58534d717ce4d0d
6796 F20101118_AABBTD interliggi_k_Page_059thm.jpg
610aca61d1c988e43e6458937c8ba842
014a352f9016c12cd294892810522492a76bccb7
87178 F20101118_AABBSQ interliggi_k_Page_014.jpg
659b328777257b697979829d34cb9f4c
5e20ad5ad6d99a1b034157a6e0130909114c564a
3875 F20101118_AABBTE interliggi_k_Page_047thm.jpg
b0d9e6a85ef2c6d3da67ee5909cd2761
8f3e094baf1f9e8f8f99e557bb2884d82d7bc40b
15524 F20101118_AABBSR interliggi_k_Page_107.QC.jpg
7f59b611852cc526a7ad56dbe6758a00
26120110efc1e7a33dfb19472dbc2e12df752d14
53637 F20101118_AABBTF interliggi_k_Page_026.pro
738c3db069480b12fa319923367dd729
4f5a18e1dc5eba41c7a8e14a22f6695451d69d40
12073 F20101118_AABBSS interliggi_k_Page_112.QC.jpg
5522ee493f082630a6ec7ad698f445b4
34c7357672fcba9b6e80b01185e7c383c3918ce9
F20101118_AABBTG interliggi_k_Page_023.tif
2133e914373ef1cbf58eada9e2a82fb3
ef1973e46c8559e7188d353880f1bb53ca774629
F20101118_AABBST interliggi_k_Page_112.tif
23cee4220ff1262aefe9247b67009821
33beab2986d33946a3308bee705a3de642cb4041
50657 F20101118_AABBTH interliggi_k_Page_035.pro
a6568a40becd0aef2ac03ccacce89c48
2b715abecff75e6a59c81f1ec91c11cd65dc2616
F20101118_AABBSU interliggi_k_Page_091.tif
4d1f529ce21628245cecaf77e0a63336
b24d0f9cff9090987b8bf0ae614d63a0f76f0e93
25670 F20101118_AABBTI interliggi_k_Page_037.QC.jpg
93470a8e92f1398526295e90a47b4c3a
0582890ac2c461db96c6dcc21cd636b31251197e
F20101118_AABBSV interliggi_k_Page_136.txt
a2264bd088db59751f917899dac2029a
f89aeda1bbc1e5131f3742b2a6a61e606706c45d
14362 F20101118_AABBTJ interliggi_k_Page_109.QC.jpg
ad39a540d964316f328b95e22dd54db3
33d80e3a34a8d62cd3b339cb5edd03119b506792
30030 F20101118_AABBSW interliggi_k_Page_138.QC.jpg
7b67f7dddee093d484129479321ec3f8
8579ef0cf308772432209bd86267c4098e3cb931
F20101118_AABBTK interliggi_k_Page_104.tif
1878955902b1529ca43a0b07537b4cd2
129bf2b049350132bd0bc8567b1101cb032344e5
F20101118_AABBSX interliggi_k_Page_022.jp2
ce15163ab70089e07e6d62aab5e876bb
70cf7c1779472b930966af6180ad1c05ad2de3e9
76521 F20101118_AABBTL interliggi_k_Page_050.jpg
eb4c02905ba5ffe7ca14b9ff7f3bc490
9f3cf206d75a4447b5e840ed7de1fe83630a87b7
2496 F20101118_AABBSY interliggi_k_Page_054thm.jpg
e3b6d37d5025c2419f0f71cf36553bd4
62f6d332f23ca1d12948222d1cf67aa92c532932
56776 F20101118_AABBUA interliggi_k_Page_097.pro
d9be2966afb6e53343c92ddb6ad8d66c
243a4f9cf3a0d839502ac857488e92e2f02b4618
929282 F20101118_AABBTM interliggi_k_Page_099.jp2
71f877b254f405ccb3ce2e6de7a17d90
00de55eea20192e74d0205fd7373bfcdb11fdbae
54928 F20101118_AABBSZ interliggi_k_Page_038.pro
6d9fa4ae2a84bc0c8b538d0f9115aa9e
37ce17cbaae43c50a425b3592aeb222673c9da61
1938 F20101118_AABBUB interliggi_k_Page_088.txt
6a17a385cfccdf7132ed8ee1309e7649
2ff3230d14fa71f0925e6e6273aeaab0251f8e35
6622 F20101118_AABBTN interliggi_k_Page_089thm.jpg
8c61aaa3b953732313bb6d2222499ff1
b627afaebf276bb33ab9b97ce0e598c75c54d423
50731 F20101118_AABBUC interliggi_k_Page_066.pro
63ee63bbd2379718027b9856b7801796
f296d3aab6d2bf280ddc3f6999430a63def39d77
F20101118_AABBTO interliggi_k_Page_021.tif
5784255a0c16ac226ef21d64dafc0d00
6cef9d3c60215bf156bd9f0a86800b9f8d47e72a
F20101118_AABBUD interliggi_k_Page_001.tif
f91416ba07a85360bfd9aa67913e947c
29a793547b0d68b16f8aff3fd3b62dc7ae15cf71
4109 F20101118_AABBTP interliggi_k_Page_104thm.jpg
344578d669fbc158506b66a044848a3e
8b58ffec40bbcec0d91b1d9d27ab166d9a5d217c
27233 F20101118_AABBUE interliggi_k_Page_117.QC.jpg
311a5b12a660c10523855e40a624aad2
484e1d98396b73673c6a009a5c91dbd8266d021b
F20101118_AABBTQ interliggi_k_Page_129.tif
465dcbbeb7b1632e61bcebd816b953dc
9f6463dc598271b1c6f4e250f9380253ed2f6df8
344486 F20101118_AABBUF interliggi_k_Page_061.jp2
99df0844409f3042a0ebde5a42113223
b2f6f4e3be538bf450aab4f1392c5db985464428
6411 F20101118_AABBTR interliggi_k_Page_036thm.jpg
6a1665f7499d45555caa4ef6180a9569
18adfb4b940839d77b8bd1506b4873e154707f0a
50762 F20101118_AABCAA interliggi_k_Page_034.pro
39e026f69a2f4a74605b894d8c249979
dd3df6499d6dbc6b20d82d4228d14e176b1bed57
628405 F20101118_AABBUG interliggi_k_Page_102.jp2
f50845f8fdfe16df4a8a472053d8dede
5f005a9a41a1ec48f9afcbdcf277ad0b4e2764eb
16787 F20101118_AABBTS interliggi_k_Page_046.QC.jpg
a832254b4bd5abfde0c66d2d1f60a84d
72c2cd43f29b35ae4d2b181387bae3c9f5388264
27828 F20101118_AABCAB interliggi_k_Page_036.QC.jpg
0a2f641a79b31480983493335b7281ac
4f40e28ce44886671475b2d2ebd3734edecf3338
16800 F20101118_AABBUH interliggi_k_Page_082.QC.jpg
58eefa4fca1c1413a2b8199d5b1e09f0
18dd98a606f2bb365c51db08bf46e297ab0574a8
860 F20101118_AABBTT interliggi_k_Page_084.txt
febaedc87eea31b946f542895723de93
a8b278b3364498690ec2c3aed425b2b163d3d780
28800 F20101118_AABCAC interliggi_k_Page_043.QC.jpg
f509ec3b005a05ed278f4b3bcf9f8db8
6e9b80d8972f76e820acb84a77f0e370fcef3ef1
27706 F20101118_AABBUI interliggi_k_Page_024.QC.jpg
218d6506ebfe9a3c3e99c0582da2eeea
e2e5313bec027714667ca6dfa11209a9041e2a8b
29390 F20101118_AABBTU interliggi_k_Page_092.QC.jpg
fe5cdc02cf7695843a4f9b572cb7ff7d
0e166f5f4ca0c6315db611603311350815e5f951
83675 F20101118_AABCAD interliggi_k_Page_069.jpg
d99dcd38c6be88546683b6436e369a2d
a03b7ae63d2599eebb9d8bf1a1ffd9b0d66be2eb
29230 F20101118_AABBUJ interliggi_k_Page_137.QC.jpg
2ef755e2d9c88fd088c79466ae4ddaad
080b0226986952a91fa1dc35d339bd3357834469
742362 F20101118_AABBTV interliggi_k_Page_056.jp2
a282a59ae8789697c6c23c93d03b41c9
7dcf81e6c0805c36592dffbb6ecf21c7b41b9124
F20101118_AABCAE interliggi_k_Page_046.tif
34aba0328138aae68ca0681e73cfb02b
9e770c98607b76ada2537c414a23408e54d08f45
24547 F20101118_AABBUK interliggi_k_Page_019.QC.jpg
aaab9da64a32af105a20acd962be6959
1dd4ff49beafbbd2b85095ae71a058c7948cb6cf
66592 F20101118_AABBTW interliggi_k_Page_146.pro
aeb2ab64777f55add2e12922af537c1d
6ab7211444ebe185f4c334b0ce2971db80625129
4241 F20101118_AABCAF interliggi_k_Page_075thm.jpg
263c6bb5af229b9defee6d7f20e8e53f
3ef494a0f409670334174ea7c4df47edec9cb2b9
3627 F20101118_AABBUL interliggi_k_Page_100thm.jpg
bc6701682f37158131b3892cf2005474
49f69d1f33be735def7e89a4b752e4fcd4d86b80
18771 F20101118_AABBTX interliggi_k_Page_141.QC.jpg
3f3dd419b0a2a085bdcbabe74586efa8
f02671ebeed4175f2e31cce452a18abfa1a04683
F20101118_AABCAG interliggi_k_Page_051.tif
a632eb0d153f4d568b4715fd6b68393a
f283e1f2938e0815ebf980622955aa1a3eab944c
F20101118_AABBVA interliggi_k_Page_063.tif
b62ea1b31dd07de003308d8ab25bcab2
b5a1e6a0e7f7b2d1bcc6d06962e5947f5e4cda9e
F20101118_AABBUM interliggi_k_Page_043.tif
29117e097d01fc967d98a9655f2d9a5a
f40916acfc0d5e4ea7a06373d5916fcd8e93c468
53211 F20101118_AABBTY interliggi_k_Page_117.pro
7387235742d7672b3b656fa2fb19233f
a42deac496852cbf1ac6a3dd55ae36f2caf9f5c2
87504 F20101118_AABCAH interliggi_k_Page_095.jpg
6689bf2e2545deb976763826d66aa3b6
d2671c8c3c2b887cbc408c8515a638d94300568b
F20101118_AABBVB interliggi_k_Page_010.jp2
aca2ab0b2222f4906f0b8d6f9022cc2e
941a87b1a3738358ac0f145cf482e23ee8919a3a
F20101118_AABBUN interliggi_k_Page_132.tif
56f63da6cb0a960a2807b1c697bb5515
263fb1fa948c0ac5409c780df3aae6256b39921b
598988 F20101118_AABBTZ interliggi_k_Page_122.jp2
e01b32b72e88814117c97b8ea9993c9f
7ca3a6995ea5e1fb450356249309d31ded70315d
1833 F20101118_AABCAI interliggi_k_Page_116.txt
482d86b1d00519e375a35e5c63a7cee3
592e9e7ec2e5fd0f5b14a840bc898d126ebc50d8
F20101118_AABBVC interliggi_k_Page_153.tif
4c9785968619a489592613b819f6bfa0
8ed29cfc5d8f7b332d7105ca8e0e40c6e1f751fb
3233 F20101118_AABBUO interliggi_k_Page_110thm.jpg
89a581677ad7ac4a6c78258a42c957a9
b09d8b013890a48cb22bc95975aad3f111fd2100
52529 F20101118_AABCAJ interliggi_k_Page_016.pro
436d1ba27935a48218f217aa0dbdeedc
0bff3f4920e402d26b65fe4ba511732cec4a7cd2
1949 F20101118_AABBUP interliggi_k_Page_056.txt
12a12e9ee48575fd47869a8fac3cd497
2501304b31fdf2c27b5b06b2fdd2f142da4ce683
F20101118_AABBVD interliggi_k_Page_110.tif
026f1761cc627884bdd409bceaa88d46
b482e1296798351491c8ddb5fd6ad771d6fe68ae
26177 F20101118_AABBUQ interliggi_k_Page_079.pro
86133002335d1c018b6365a45417a94d
1b63d2b7e7f8cb24a8d5173b9f8c477e66bb7254
534405 F20101118_AABCAK interliggi_k_Page_031.jp2
24e91025b55757050297dc2f8ebf124a
2c77da993ac0aec0cd9d44f8999bc6bf588ac811
63632 F20101118_AABBVE interliggi_k_Page_151.pro
dab85d36fe3acc05d145c468049e9fef
b7e9ad5a4faf7c63d979401e60992c22a95716c8
31486 F20101118_AABBUR interliggi_k_Page_031.jpg
09343dff956f8d85c345c84fee7f290f
a7f9539dd9e70bdb74ce7f4ab1915e041ab0f8ef
88860 F20101118_AABCBA interliggi_k_Page_034.jpg
aee6ba2d5d017464f7ee98b0815a11bf
170464697d692773a0d81647f7dc1ebe52ad7f71
3362 F20101118_AABCAL interliggi_k_Page_013thm.jpg
581d0e27c47b9a37e484b16c12990432
5e787a80a79bf280ce5875201f1eafb26ad33f0c
F20101118_AABBVF interliggi_k_Page_045.tif
d5391fea328d34429f4e9a09527b4c09
b250ea1f952c738dfffafe753b0f952e3cbb11ab
87409 F20101118_AABBUS interliggi_k_Page_133.jpg
3ffeca5f50fc2c75bd7d9e5a96ef3325
f34f749d820e0421b5882a9a9669a873040ab82b
F20101118_AABCBB interliggi_k_Page_036.tif
c4f455a33b722df2ecf11c6b421488b8
84a57989711952a644b4f1c25af2029963201307
27980 F20101118_AABCAM interliggi_k_Page_072.QC.jpg
08edb89202f949730b236e899fd314dc
1d49cfac3f75ab78ccf1ee89166751148ac8c071
87858 F20101118_AABBVG interliggi_k_Page_041.jpg
5daeb8dc9b0b73595014be6e61c3bc00
12693bffc478e4f7a6cf25663dcaab048ba9b205
1908 F20101118_AABBUT interliggi_k_Page_020.txt
817d822044459b8e77a5f5f52bb7f8dc
a7fc577cf9031142269629b50ade132be0d9d6b5
94215 F20101118_AABCBC interliggi_k_Page_138.jpg
0255483ece4676fe0d2955b53dd2969f
080e89c2396f8ade11f48272ca9d942dae5e52d9
12482 F20101118_AABCAN interliggi_k_Page_154.pro
2cff730056e708213506bf618b62add1
b0852d4cd78114d60c52184374547846bcda2104
F20101118_AABBVH interliggi_k_Page_067.tif
263e4a0ded3c1a61c9805a15f07bd722
f4af6b8fc952ef432b67ade1bc3871571235e837
39970 F20101118_AABBUU interliggi_k_Page_112.jpg
99bb918425ba85c64e766c867331f1f5
bb6148fbc6628021e8aeb787b5c72bc15b36c2a2
27001 F20101118_AABCBD interliggi_k_Page_068.QC.jpg
bd74559e111ecea735dc9f6c7f717e44
02a6e68fa363061c515496a751ae32f9e19e43cc
718027 F20101118_AABCAO interliggi_k_Page_128.jp2
cf09d91e721efca6eb0c2a6d2cdf4eb3
7a76aa16455aef95ad6a2fe27751d8c1ab2d86ce
25766 F20101118_AABBVI interliggi_k_Page_154.jpg
7494d1f49073f1de13fc2f0e347d2bb2
2cfc8de811d741dd2a5b17d2c3f99b09c6dd0aa6
F20101118_AABBUV interliggi_k_Page_067.jp2
9bd1baf3d3222a43b166b85247089e64
6f8abda11c6044cd00b91beb38423cfcb6f1dc7d
2206 F20101118_AABCBE interliggi_k_Page_093.txt
077450a57ac9b5896a5df88513b5415a
c260e3b09925cb23003001c0425d9f6eb6c87b32
47034 F20101118_AABCAP interliggi_k_Page_124.jpg
47ffef6070695ada207ba233cf05de0d
8e36dce627ba89037f3feed87fa4ee9247a3a24a
F20101118_AABBVJ interliggi_k_Page_094.tif
4c9de97ab3dfb2c235d026d546e1d50a
07ddd55ce2c3dc1221e3eeb2a5e255a6d4e8c6b3
16637 F20101118_AABBUW interliggi_k_Page_077.pro
8beb3dd30374bde7339d8255b0761609
d4635fca3836bc0f4cedae60901f6916e88a89e4
30178 F20101118_AABCBF interliggi_k_Page_147.QC.jpg
5b533836f673e46ba8f9bb4a545201c2
f462189d2e158b1171be568531cd68f91c03fe85
80474 F20101118_AABCAQ interliggi_k_Page_086.jpg
715649a3e8c8036fb973da569c0c9056
b862dfe4fdceadd72879219c1ca268d1935c7abe
6170 F20101118_AABBVK interliggi_k_Page_067thm.jpg
8165a358ce935e4a8e4f6dd05bf43d61
31697e39e7a006270f92012f4d1c0531db60cca2
83775 F20101118_AABBUX interliggi_k_Page_101.jpg
1e3dec31a6b7aa7f097b61a643786d52
4846d879b65480830c057c3e94ae3ca6367d4a7d
6000 F20101118_AABCBG interliggi_k_Page_101thm.jpg
58b4bc6752e1f294130a91ff0c0bdd31
88446911f79baa724722122a39d1006f2ca5ebc3
54599 F20101118_AABBWA interliggi_k_Page_135.pro
577acf1de2e276226c59781743d40c26
bc94e0146496d1c39e856e42ff24cfe55914a54a
F20101118_AABCAR interliggi_k_Page_133.jp2
85f12b3897b3762d13b271edd324a056
511ca24382fcba18533f004f648c8e23f21b5f41
27553 F20101118_AABBVL interliggi_k_Page_021.QC.jpg
3b8d86835c180527b629a8519fc6ab1a
a822b089cef444db346c9ba055671aa33bed4f06
28223 F20101118_AABBUY interliggi_k_Page_044.QC.jpg
5cd322e93d4c29981a57cc8dbbb77ecf
f802bf1d20c673db6ceaa2cdc0c209b1818d130e
50638 F20101118_AABCBH interliggi_k_Page_028.pro
1c7d7a9decaf7fbc5cf55677913e8b8a
12c2e04e30d0348ff3eac92386eaece9bac6884c
F20101118_AABCAS interliggi_k_Page_037.tif
c77f167d31ed7d934fb7e8a4facd5cb7
c0f83d2953218b6daecdc11ea91437c0539698f1
F20101118_AABBVM interliggi_k_Page_139.tif
9f6d8e4d5957275ca775dd6682a18bcc
e2508f0470533481ef8a25b42c6a5ab0d9cde841
F20101118_AABBUZ interliggi_k_Page_026.tif
64c1a42e7b82ea0ed5cf69c14de4e1fa
e86f843063f281d3f72d7e0316dc962875ce6c42
42941 F20101118_AABCBI interliggi_k_Page_102.jpg
8d288dde31527f8e0688dabc674aed02
cbcd588ba58fe5d4e25df94565f9a1e693d111cb
200 F20101118_AABBWB interliggi_k_Page_003.txt
01d762e71d31bd5285aac86776261a98
47a833d8db84467f26873632c1ddf9bb24a41390
93557 F20101118_AABCAT interliggi_k_Page_027.jpg
fd1ea16f006842c5c189f784bcc98722
83bbee3f3d84f4e536984a11c6bfa5827e645e17
F20101118_AABBVN interliggi_k_Page_119.txt
ee40a24865111cbd2236066e265bf601
95a2799aa34090f73bbf596df0e97a35a588f92b
480109 F20101118_AABCBJ interliggi_k_Page_076.jp2
53837911fbbb93ead1c9b4aae14e11c1
718ba3bb832d01a020e313ae91695ab91fe795b2
18443 F20101118_AABBWC interliggi_k_Page_104.pro
b7da68708ab0c347a2804b03d9541c8d
33d18258481b6385be0f0829d0dcdd6d1384f66d
44688 F20101118_AABCAU interliggi_k_Page_013.jpg
833b48c677f86d9e70afc831581ad191
01151b451e084459a0caec0085ff9774de786aea
F20101118_AABBVO interliggi_k_Page_136.jpg
53d9f4b1682dbf321e766c7fe914a2c1
e6b09b4a150680360b57316fb2dc9a73d664a77f
F20101118_AABCBK interliggi_k_Page_008.jp2
6010e93e665c5be5db11340dd373bac1
08e06a1fffdcb7ec69522c61c607354aae4a1462
933291 F20101118_AABBWD interliggi_k_Page_144.jp2
93388516816f07a4282846f27ed00250
e4dc460b58b4227febecd06904d5404d45b35a9f
F20101118_AABCAV interliggi_k_Page_019.txt
9c75d880c08fdb9d3ccaa78616aaa770
ba4ce2a94bbbfc59ac587d44081d95f794813740
1051967 F20101118_AABBVP interliggi_k_Page_146.jp2
628e9b2a9417fd6bb3d379bd909444f2
b400acff4982052ba0235e59834f09ff420d2966
F20101118_AABBWE interliggi_k_Page_145.jp2
5bd1e60d464302e9291fa3778979aa6b
8b0e332a07b39fb16f6560ac141383bcf76e90da
F20101118_AABCAW interliggi_k_Page_038.jp2
dbd4a5b16a5d4f035ff3692f7b4d794e
022b2a89f2dac8fa9ad92f61bdcbbe1e02fa0640
6323 F20101118_AABBVQ interliggi_k_Page_113thm.jpg
5681a8fc4703e52c86f27e35953a1f58
779ce702a7452f278c9671219de08e1ce3c118a4
3870 F20101118_AABCCA interliggi_k_Page_126thm.jpg
5e4a2292277a02f0502dbda9fcb3ca6b
d93dbe6d94cb6d05e1f2ca1dfb6de2e5bd2ce039
F20101118_AABCBL interliggi_k_Page_027.jp2
43e88e88d5e346471a8214dd76cb7e46
e6f8ebca114b2a841b497c7fcc4788059b06dd4f
18871 F20101118_AABBWF interliggi_k_Page_105.pro
5830251dc7e7ad3b822bbb03c1f9f749
7c11a93d33c70fa0444815748f4cb0eb79f9a0c4
1448 F20101118_AABCAX interliggi_k_Page_074.txt
c92404371418cc3d0d1035d602c83ac9
b9284b5c1d8b37710da6dc9b90bd4f4b05b4073b
F20101118_AABBVR interliggi_k_Page_086.jp2
631453cb878169ca91e9e03e42f5fadb
a172fd297adddcfa2f652995a86e47c520c15aa3
F20101118_AABCBM interliggi_k_Page_052.tif
0bb485c0c81310075c8abc63119038f6
4b997e9161b94061e347d655b549d9d2865fed89
196920 F20101118_AABBWG interliggi_k_Page_053.jp2
0481133a8adb46fd39a962e20060d7e7
5e60c1a3037bb381d71e6d2c3f9941828075662a
F20101118_AABCAY interliggi_k_Page_093.tif
45df45218d7d32c55704da5419071ff5
dfd940963e66bd376bce8c0e6df61603a2a7fc70
13875 F20101118_AABBVS interliggi_k_Page_124.QC.jpg
06260a943313d63eac63cee5aedfc656
9450bc7e56416276ebafb903711dda5ce24485ff
2113 F20101118_AABCCB interliggi_k_Page_042.txt
77416d1d0d1d3643d0dc2c272e8e81ea
7706ee1d259e3d572daf7fd975836d7a07da9931
6648 F20101118_AABCBN interliggi_k_Page_018thm.jpg
930c4668a3575be9e65c6a9ce12d1950
c87b2b2f0ff533696bc1caaede297facf86099da
30794 F20101118_AABBWH interliggi_k_Page_084.jpg
1cb73d7585614c1d71d602a9ff1bc17a
2b1b208f42b2a47ffecbe95d950805be4dbdfdd7
F20101118_AABCAZ interliggi_k_Page_042.jp2
a2c6a5f19f9793671ac293aa5adea491
c77304b0e95cbfea22a1f458d14899977e98001b
1051942 F20101118_AABBVT interliggi_k_Page_068.jp2
4b2e4d856fb72f28bb296dec9d8c5cf7
d007f4ae1fa20546410e4d8b3f4af9fd5afe6fee
F20101118_AABCCC interliggi_k_Page_089.tif
167a0f2eccf6b41abcef4b065c325aac
c3ffd9ecacf5faa1a22c27591991457ba9687a22
1345 F20101118_AABCBO interliggi_k_Page_007.txt
ec2e15cd97dee086e818d953cc6b3eaf
0f4fa51a470d34a1d06ac0eeed9d3de602aa6e19
2041 F20101118_AABBWI interliggi_k_Page_063.txt
f43ab190d6403074dc3b34c2597cccbb
debae4e1ecb0c292bab16dd17f83c3d01161e70e
1236 F20101118_AABBVU interliggi_k_Page_124.txt
7ad1b9342163e800d292f9b9d253b2c1
8bbf8c4181cad7dbe4cb358f88da1bffcf659258
106666 F20101118_AABCCD interliggi_k_Page_006.jpg
a213d146586cec1f8ca592c72af2fb1d
76e14ba42a5cdc8269bc2c0f49c38bfe2d13977c
55547 F20101118_AABCBP interliggi_k_Page_098.pro
517e55962d57168e9b62e13ba6b34484
1c519b2262a2c356b650eb6de4a4f5b749536bde
46693 F20101118_AABBWJ interliggi_k_Page_110.jpg
5c10e7a6438483bc24714004db56620d
06a9c26621ef1ba98ad79e7d8058074df4e629f1
88161 F20101118_AABBVV interliggi_k_Page_071.jpg
d9431f84658f66dc5b6da2b4abe98971
cf987c164e4e09f1b5a25cc79ac09233e85e1072
580689 F20101118_AABCCE interliggi_k_Page_013.jp2
c55403ab1864a81a2522ccf8673310fb
9a069edb1d3ae1ee8bfdd9357e5f73fe1292b168
5480 F20101118_AABCBQ interliggi_k_Page_051thm.jpg
bf1c6f3b6944be336b31711035c8b188
da7b9fa7d68ffa0184e83e8b7d439f972deb968a
2187 F20101118_AABBWK interliggi_k_Page_092.txt
5a0da7ac1eede824163b047f845b82ab
5d245d3059602054749160425f61703fa81a89d2
6555 F20101118_AABBVW interliggi_k_Page_137thm.jpg
c4fdb4eb9ad81c707939838c37c88b97
ee82b24b95a32ed67257ba6822b20268bdf880c7
25454 F20101118_AABCCF interliggi_k_Page_013.pro
b0c847c1b1cf312c6cbfd5829bdbcaca
b9b2123d9ffbbc87972b7191d89bd53306c86385
F20101118_AABBXA interliggi_k_Page_151.tif
4423c9a2cb6673263b2d8f4cfa7b5c49
dbbd4a6a9b370b9aa4c173018328a2f520596316
27990 F20101118_AABCBR interliggi_k_Page_035.QC.jpg
e916a8e3e6bde409101ab10463f32277
d387b6976883d141a8caf6fe4d633189ce5e53fa
2733 F20101118_AABBWL interliggi_k_Page_150.txt
399921ef8c3d7fbd33650eb55b8fc5af
0c7ba7ddfd6de21461d35ee61e5e1cbb73c8e61d
63649 F20101118_AABBVX interliggi_k_Page_148.pro
a948eaa296075afaaccccdaccbe071f2
e95a4dff1e20a0ae90444a1c0f8cb9a54fd4b241
3570 F20101118_AABCCG interliggi_k_Page_077thm.jpg
c1f413f33b8cd0e196b4d0e830f25eea
61f6f59df8f7662a2020e617e2f5983f2ae63775







CHARACTERIZATION OF ACTIN-BASED MOTILITY ON MODIFIED SURFACES FOR
IN VITRO APPLICATIONS IN NANODEVICES





















By

KIMBERLY A. INTERLIGGI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































2007 Kimberly A. Interliggi
























To my encouraging parents, Denni and Vicki Interliggi,
my siblings, Jen and Tom, and my boyfriend, Matt Crim,
for their continued support.









ACKNOWLEDGMENTS

I thank my advisor, Dr. Richard B. Dickinson, for his support over the past four years. His

guidance and encouragement have helped me succeed in many aspects of my graduate

experience. I appreciate Dr. Yiider Tseng for always expressing his interest in my research and

career and encouraging me along the way. I thank Dr. Spyros Svoronos for his dedication as a

committee member. I acknowledge Dr. Anuj Chauhan for his guidance and helpful advice from

the very beginning of my graduate career. I also thank Dr. Jennifer Curtis for her dedication to

the graduate students in this department. Finally, I acknowledge the faculty and staff in the

Department of Chemical Engineering for continuously supporting my efforts in many aspects of

my graduate school career.

I am grateful to have had the opportunity to work with Dr. Daniel Purich in the Department

of Biochemistry. Dr. Purich was involved on a daily basis with my lab work and was constantly

teaching me new skills and sharing new ideas with me. I also thank Dr. William Zeile, who

taught me many experimental and research techniques, and was always willing to listen to my

research problems and help me work through them. Dr. Joseph Phillips and Dr. Fangliang Zhang

are greatly appreciated for their companionship and knowledge in the lab. I also thank Dr.

Suzanne Ciftan-Hens and Dr. Gary McGuire at International Technology Center in Raleigh, NC

for their collaboration on this project.

I thank Dr. Adam Feinberg, who was instrumental in helping me start my project. I thank

the members of my group, who were consistently helping me to understand and work through

daily problems and always provided good company: Dr. Luzelena Caro, Dr. Colin Sturm,

Gaurav Misra, Dr. Jeff Sharp, Dr. Huilian Ma, and Adam Wulkan. I also thank Andre Baran for

his assistance and constant support.









TABLE OF CONTENTS
page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

LIST OF FIGURES .................................. .. ..... ..... ................. .8

L IST O F A B B R E V IA T IO N S ............................................................. ..... ................................ 11

A B S T R A C T ................................ ............................................................ 12

CHAPTER

1 INTRODUCTION ............... ................. ........... .............................. 14

1.1 A ctin .................... ............................................................................ 15
1.1.1 Filam ent G row th ..................... .. ........................ .. .. ...... .............. 15
1.1.2 A ctom yosin ................................................................... .... ....... .... 16
1.1.3 Actin-Associated Proteins............................................... ...............17
1.2 Experimental Methods for Actin Polymerization on Modified Substrata ....................18
1.2.1 Total Internal Reflection Fluorescence Microscopy ...................................18
1.2.2 M icrocontact Printing ............................................... ............................ 21
1.3 A ctin-B asked M otility............ ............ ........................... .. .... ........ ........ 22
1.3.1 Listeria and Particle M otility ........................................ ........................ 22
1.3.2 M mechanism s ......................................................................25
1.4 B ion an odev ices .......................................................................2 6
1.4.1 Sliding Filam ent A ssay .............................................................................. 27
1.4.2 Im m obilized Filam ent A ssay ........................................ ........................ 28
1.4.3 M material Transport.................................................. ............................... 29

2 GUIDANCE OF ACTIN FILAMENT ELONGATION ON FILAMENT-BINDING
T R A C K S ................... ................... ........................................................ .. 3 2

2 .1 Introdu action ...................................... .................................................. 32
2.2 M materials and M ethods....................................................... ........ .......32
2.2.1 Protein Preparation .................. .......................... .................... .. 32
2.2.2 M icrocontact Printing ............................................... ............................ 34
2.2.3 A ctin Polym erization ..................................... .......................................35
2.2.4 TIRF M icroscopy and Data Analysis..................................... ............... 36
2.3 R results ....... .................... ......... ...... ............. .. 38
2.3.1 Filament Binding to Stamped Surfaces................................. .................. ....38
2.3.2 Alignment of Filaments ............................. ................... 39
2.3.3 NEM-Myosin Concentration...................... ..... ........................... 41
2.3.4 Control of Actin Polymerization .............................................. ...............42
2 .4 D iscu ssion ................. .................................................................. ............................... 4 3
2.4.1 M echanism for Filament Alignment .... .......... ....................................... 43
2.4.2 Potential and A applications ........................................ .......................... 45









3 SIMULATING ACTIN FILAMENT ELONGATION ON MODIFIED SURFACES .........63

3 .1 In tro d u ctio n ............. .. ............... ............................................................................. 6 3
3 .2 M eth o d o lo g y ........................................................................................................... 6 3
3.3 R results ......... ..... .......................... ..... .......................66
3.3.1 Description of Simulated Filament Elongation....................... ..............66
3.3.2 Probability of Filament Rebinding..... .................... ..............67
3.3.3 Alignment of Filaments............ ..... ................................ 70
3 .4 D isc u ssio n ............................................................................................................... 7 1

4 ACTIN-BASED MOTILITY OF LISTERIA AND PARTICLES ON MODIFIED
S U R F A C E S .................................................................................8 6

4 .1 Introduction ......................... ................................... ............................86
4.2 M materials and M ethods.............................................. ........................................... 86
4.2.1 Listeria monocytogenes Growth and Protein Purification.............................86
4.2.2 Bead Preparation ......... ...... .................................. ... .. ..................... 88
4 .2 .3 M utility A ssay ............... .............................................. .... .... .... .....89
4.2.4 Fabrication of Channel Devices................................................... ............. 89
4.2.5 M icroscopy and A nalysis............................................ ........... ............... 90
4 .3 R results ..................................... ................................. .. ....................... 9 1
4.3.1 Confining Particle Propulsion to the Surface.......................... ................91
4.3.2 Effectiveness of NEM-Myosin Surfaces........................................................92
4.3.3 Particle Velocity and Tail Characterization.................. ...................... ...............93
4.3.4 G uiding Particle Propulsion ................................... ............................ ......... 95
4.4 D discussion ........................................... .................... ......... 96
4.4.1 Mechanics of Actin Rocket Tails on Surfaces ............................................96
4.4.2 Biochem ical Considerations ........................................ ........................ 97
4.4.3 Considerations for Bionanotechnology ................ ...................... ......... 98

5 SINGLE FILAMENT ACTIN-BASED MOTILITY OF PARTICLES .............................113

5 .1 Intro du action ...................................................13..........
5.2 M materials and M ethods......... .................................... ........................ ............... 114
5.2.1 Protein Preparations ....................................... ...............................114
5.2.2 Bead Functionalization .....................................................................114
5.2.3 M utility A says ........................................................... ........ ... 115
5.2.3.1 A attached beads ........... .... .................... ........ ... ................... ... .. 115
5.2.3.2 NEM -myosin surfaces ........... .................................... ............... 116
5.2.4 M icroscopy and Analysis................................................... .. ................. 116
5 .3 R e su lts ............................................................................................1 1 6
5.3.1 Actin Asters ................. ........................116
5.3.2 Single Actin Filaments .............. .............. ........................... 118
5.4 D discussion ........................................................119






6









6 SU M M ARY AN D FU TURE W ORK ..................................................................................133

6 .1 Single A ctin F ilam ents ............................................................................... ........ 133
6.2 A ctin-B asked M utility ........................................................... ............................ 135
6.3 Recommendation for Future Work ...................... ..................136
6.3.1 Filam ent-B finding Tracks ......................................................................... 136
6.3.2 Three-Dimensional Surfaces for Larger Structures .....................................137
6.3.2 Use of End-Tracking Motors ............................ .......................... 138

APPENDIX: MATLAB CODE ............................................................ ................... 140

L IST O F R E F E R E N C E S ............................................................................. ..........................145

B IO G R A PH IC A L SK E T C H ......................................................................... .. ...................... 154








































7









LIST OF FIGURES


Figure page

1-1 Treadm killing of an actin filam ent ............................................. ............................. 30

1-2 Experimental set-up for binding filaments to a surface ..................................................30

1-3 Schematic of microcontact printing protein on glass .....................................................31

2-1 Images of microcontact-printed myosin tracks on a glass coverslip .................................47

2-2 Time-lapse image of actin filaments on a BSA-stamped surface..............................48

2-3 Total internal reflection fluorescence microscopy images of elongating actin
filam ents on N EM -m yosin tracks ............................................ .............................. 49

2-4 Filaments in BSA region undergo large thermal undulations................ .............. ....49

2-5 Total internal reflection fluorescence microscopy images of undulating ends of
elongating film ents ..................................... .................. .............. ......... 50

2-6 Filament elongating passed the track edge at small .......................................... ........... 50

2-7 Filament alignment as a function of filament density on tracks.....................................51

2-8 Effect of track width on filament alignment ..................................... ........ ............... 52

2-9 Histogram showing the fraction, of filament ends that rebind to tracks after their
elongating ends cross track boundaries ............. ................................... 54

2-10 Scatter plot showing each segment alignment with the track edge as a function of the
distance from the track edge ........................................... .................. ............... 55

2-11 Dependence of filament alignment and elongation rate on the concentration of
N E M -m y o sin ............................. ............................................................... ............... 56

2-12 Scatter plots showing the alignment of individual filament segments ............. ..............57

2-13 Filaments accumulate in the BSA region of the stamped surface. ................................59

2-14 Incubation tim e oflyophilized rhodam ine actin ...............................................................59

2-15 Increasing the concentration of profilin visibly decreases the density of actin
filaments on NEM -myosin treated surfaces................................... ........................ 60

2-16 Elongation rate of actin filaments as a function of profilin concentration ........................61









2-17 Illustration of the likely mechanism for actin filament alignment on NEM-myosin
tracks .......................................... ...... ............................................ 62

3-1 The x- and y-positions of elongating filament ends as a function of time......................75

3-2 Effect of change in length (step-size of simulation) on the filament rebinding
probability ................. .................................... ...........................77

3-3 Effect of the number of modes on filament rebinding probability .............. .......... 78

3-4 Effect of total filament length on filament rebinding probability .............. ...............79

3-5 Effect of binding probability constant, Kp (tm-1xsec-1), on filament rebinding
p ro b ab ility ............................................................................. 8 0

3-6 Effect of persistence length on filament rebinding probability. .................................81

3-7 Effect of track width and number of modes on the alignment of filaments .....................82

3-8 Effect of track width and binding probability constant on the alignment of filaments .....83

3-9 Effect of binding probability on the alignment of filaments................ ............... 84

3-10 Effect of track width and persistence length on the alignment of filaments.....................85

4-1 Listeria and 500-nm diameter bead propelled by actin rocket tails..............................100

4-2 Rotation of a helical actin rocket tail in solution............ ..............................100

4-3 Listeria rocket tails on NEM-myosin and BSA-treated surfaces...................................101

4-4 500-nm diameter beads attached to rocket tails bound on NEM-myosin and
BSA-treated surfaces ................................... .... ... ...... ..............101

4-5 Fields-of-view with large percentage of tails bound to surface ............... .................102

4-6 Fraction of actin rocket tails bound to NEM-myosin and BSA-treated surfaces. ...........103

4-7 Change in x- and y-position over time for beads on NEM-myosin and BSA surfaces ...104

4-8 Helical actin rocket tail confined to NEM-myosin-treated surfaces.............................104

4-9 Actin rocket tails in total internal reflection fluorescence microscopy .........................105

4-10 Magnified image of actin rocket tail with protruding filaments.................................... 106

4-11 Actin tail elongation on NEM-myosin and BSA surfaces....................... ...............107

4-12 Average tail elongation rates determined from the slope of a best-fit line................108









4-13 NEM-myosin surface effect on persistence of tail................................109

4-14 Stamped surfaces with 500-nm diameter beads attached to actin tails.........................110

4-15 Actin tails bound to NEM-myosin-treated exposed glass of fabricated device .............. 11

4-16 Actin rocket tail encounters CYTOP wall .................................................................... 112

5-1 Growth of actin filaments/bundles on 50-nm diameter beads ....................................... 123

5-2 Fluorescent intensity of actin filam ents/bundles.............................................................124

5-3 Actin asters recovery after photobleaching.............. ............................. .... ............. 125

5-4 Enlarged image of asters reappearing after photobleaching .......................... .........126

5-5 Fluorescent intensity of photobleached filam ents .........................................................127

5-6 Recovery rates and equilibrium intensities of photobleached filaments .........................128

5-7 50-nm diameter beads bound to surface with single filaments or bundles...................130

5-8 50-nm diameter beads in solution on NEM-myosin surfaces .............. ... ................131

5-9 Actin motility assay with and without beads on NEM-myosin surfaces.......................132









LIST OF ABBREVIATIONS

ADF Actin depolymerizing factor

ADP Adenosine diphosphate

AFM Atomic force microscopy

APES 3-aminopropyltriethoxysilane

Arp2/3 Actin related proteins 2/3

ATP Adenosine triphosphate

BHI Brain-heart infusion media

BS3 Bis(sulfosuccinimidyl suberate)

BSA Bovine serum albumin

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

EDTA Ethylenediamine tetraacetic acid

EGTA Ethylene glycol tetraacetic acid

EM Electron microscopy

HMM Heavy meromyosin

NEM N-ethylmaleimide

PDMS Polydimethylsiloxane

PMSF Phenylmethanesulphonyl fluoride

TIRF Total internal reflection fluorescence

VASP Vasodilator-stimulated phosphoprotein









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CHARACTERIZATION OF ACTIN-BASED MOTILITY ON MODIFIED SURFACES FOR
IN VITRO APPLICATIONS IN NANODEVICES

By

Kimberly A. Interliggi

December 2007

Chair: Richard Dickinson
Major: Chemical Engineering

The cytoskeletal protein actin generates forces for various processes by polymerizing into

filaments. In vivo, actin works with the motor protein myosin to produce muscle contractions

and with proteins acting as end-tracking motors responsible for cell and bacterial motility, such

as the motility ofListeria monocytogenes. End-tracking proteins bind the polymerizing end of

an actin filament to a motile surface, creating persistent attachment during filament elongation.

Both types of motors use the energy from ATP hydrolysis and can be exploited in vitro in

bionanodevices, which require forces to transport objects on a micro- or nano-scale, possibly

against flow or diffusion gradients.

Our study has focused on the guidance of single-filament elongation and filament bundles

(rocket tails) to orient elongation in vitro. Microcontact printing, a technique that stamps protein

patterns onto glass surfaces through adsorption, was used to create filament-binding tracks of

modified myosin (void of its motor activity), which successfully bound and guided single actin

filament elongation in a manner dependent on track width and surface conditions. These results

confirm the capability of this method to be used for the motility of objects attached to single

actin filaments and for the creation of immobilized tracks of actin filaments for myosin-mediated









transport. Simulations were used to characterize the system further and have the ability to help

make predictions for other types of filaments and systems.

Modified myosin surfaces also confined actin rocket tails attached to particles and bacteria,

reducing the Brownian motion of the motile objects. Channels formed through

photolithographic techniques on glass surfaces were used to attempt to guide these particles.

Single actin filaments attached to smaller particles were also characterized to determine the

potential for single-filament propulsion in nanodevices. We conclude that actin filament-binding

proteins can be applied to surfaces using adsorption and microcontact printing and that this

technique is effective in binding and guiding filaments in various systems, including single and

bundled filaments. We predict this technique can be applied to other systems undergoing

actin-based motility, making it a versatile method for bionanotechnology.









CHAPTER 1
INTRODUCTION

Muscle contraction, cell movement, intracellular bacteria motility and cell division are all

processes that involve biomolecular motors, a group of proteins that utilize the energy from

adenosine triphosphate (ATP) hydrolysis to do work on a system (1). These biomolecular

motors can be exploited for the in vitro transport of nano-cargo (e.g. beads, bacteria, DNA) (2-7),

as well as in biosensors, microfluidic devices, and micro- and nanoelectromechanical systems

(MEMS and NEMS) (8-15). One approach has been to pattern surface regions with a

high-affinity for the adsorption of the molecular motors kinesin and myosin to guide transport of

pre-assembled microtubules and actin filaments, respectively (8, 15-21). Other methods rely on

surface-immobilized microtubules and actin filaments to create tracks for kinesin-mediated (3, 5,

22) and myosin-mediated particle transport (6, 23, 24).

Current research has been aimed toward understanding another type of biomolecular motor

that couples force-producing, polymerizing filaments to a motile object (25-27). These motors,

known as filament end-tracking proteins, are thought to take part in the formation of

lamellapodia and filopodia during cell crawling, propulsion of bacteria, such as Listeria

monocytogenes (28, 29), and chromosome separation during cell division (30). End-tracking

motors provide a versatile mechanism for force generation in vitro, including the potential to be

used as micro- and nano-scale actuators that can transport objects against obstacles, including

opposing forces.

Both myosin and end-tracking motors provide many advantages to bionanotechnology.

Nanofluidics is one method for high-throughput analysis of biological material, but due to the

high surface to volume ratio, friction can cause significant losses. Molecular motor systems can

transport material without flow, therefore eliminating friction losses (10, 31). Other applications









of biomolecular motors in nanodevices are broad and include biomaterials that no longer depend

on equilibrium conditions for effectiveness (10), extremely sensitive and fast single-molecule

detection (32), and nano-scale information storage and processing through surface-probing (33).

Both surface modifications and biochemical parameters play a key role in the exploitation of

biomolecular motors. We have focused on using suitably patterned surfaces to place and guide

elongating actin filaments or bundles of filaments attached to particles.

1.1 Actin

Globular actin (G-actin), a 43,000 Da protein with a radius of 2.7 nm, is a cytoskeletal

protein found in eukaryotic cells. It has a deep cleft for binding ATP or adenosine diphosphate

(ADP) and an associated cation (Ca2+ or Mg2+) (34-37). G-actin can polymerize into filaments

(F-actin) under conditions of high ionic strength in vitro, producing a 7-nm diameter

semi-flexible, double-helical filament with repeats every 37 nm (1, 38). Within the filament,

actin monomers align with all of their clefts pointing in the same direction (toward the (-)-end).

This asymmetrical orientation causes polarity in the filament, with a faster-polymerizing (+)-end

and a slower polymerizing (-)-end.

1.1.1 Filament Growth

Actin polymerization includes a nucleation step, followed by filament elongation and

treadmilling (39). Nucleation requires that three ATP-actin monomers come together to form a

polymerization nuclei. In vivo and in cell extract systems, actin-related protein 2/3 (Arp2/3)

complex has been experimentally confirmed to be located at the site of actin filament assembly

and assumed to serve as a polymerization nuclei (40, 41). As its name implies, Arp2/3 has two

subunits with similar structures to actin (42). Elongation occurs when ATP-actin monomers add

to the filament (+)-end, followed by hydrolysis of the monomer-bound ATP molecule during or

slightly after the addition of the next monomer, creating ADP-Pi-actin monomers within the









filament. The Pi is released, forming ADP-actin monomers near the (-)-end. The hydrolysis

results in a (+)-end rich in ATP-actin and a (-)-end rich in ADP-actin (36). In vitro, the (+)-end

has a lower critical concentration than the (-)-end (0.1 and 0.6 riM, respectively (43)), and when

the concentration of free G-actin in solution is between these two values, a behavior known as

treadmilling occurs (Figure 1-1) During treadmilling, the filament polymerizes at the (+)-end

by the addition of ATP-G-actin and depolymerizes at the (-)-end. Once free from the filament

(-)-end, ADP-actin releases its nucleotide and binds to ATP, replenishing the monomer pool for

(+)-end elongation and starting the cycle over (44-46).

In vitro solutions of F-actin have been found to contain filaments with lengths of tens of

microns (47). Filaments undergo Brownian motion in solution, which can be modeled based on

filament length, L, and persistence length, k, which is the characteristic length over which

thermal undulations cause the filament orientation to be uncorrelated. For actin, X has been

found to be between approximately 10 and 20 [im, depending on the conditions (48, 49).

Because X and L are on the same length scale, F-actin is considered to be a semi-flexible

polymer. The thermal motion of phalloidin-stabilized filaments has been quantitatively

described by using the bending energy of a rod with both ends free and then performing a normal

mode analysis of the bending excitations (49).

1.1.2 Actomyosin

The actin/myosin complex (actomyosin) is a type of biomolecular motor that is mainly

responsible for the contraction of muscles but also plays a role in actin filament dynamics in the

cytoskeleton. Myosin, a 500 kDa dimeric protein, has two heads that interact with an actin

filament and through the hydrolysis of myosin-bound ATP, move along the length of the

filament. The hydrolysis of ATP to ADP causes the entire head region to bend in a hinge-like









manner due to local conformational changes near the nucleotide binding site (50). ATP first

binds to myosin attached to an actin filament, causing the actin filament to immediately

dissociate. The ATP hydrolyzes to form ADP-bound myosin, changing the structure of the

myosin head from bent to extended and allowing for the myosin to rebind to the actin filament.

The ADP is released, causing the myosin head to bend at the hinge (known as a power stroke),

moving the actin filament 110 A (51, 52). Myosin then binds to a new ATP molecule and the

cycle repeats.

1.1.3 Actin-Associated Proteins

The cytoplasm of cells contains approximately a concentration of actin 600 to 1200 times

the (+)-end critical concentration of purified actin. Therefore, the cell uses various actin-binding

proteins to regulate actin polymerization and depolymerization (53). Two of these proteins

include thymosin-p4 (5 kDa) and profilin (15 kDa), which both bind with higher affinity to

ATP-G-actin than ADP-G-actin (54, 55). Thymosin-p4 sequesters actin monomers and prevents

them from adding to filament (+)-ends while profilin aids in polymerization by catalyzing the

exchange of ADP to ATP on G-actin and ushering ATP-G-actin to filament (+)-ends (56).

Profilin also prevents G-actin from forming nuclei, which can control the density of filaments

(57).

Arp2/3 complex, as mentioned previously, increases the nucleation rate of filaments. In

addition, it plays a key role in the development of actin networks. Arps are responsible for

branching of filaments by binding to the side of actin filaments or filament (-)-ends to nucleate

daughter filaments that extend from the mother filament at 700 (58). Another important group of

proteins is actin cross-linking proteins, such as a-actinin, which help to create various actin

structures and networks by connecting filaments (59). Actin depolymerizing factor

(ADF)/cofilins (15-20 kDa) bind with high affinity to ADP-F-actin, causing an acceleration of









the depolymerization of filaments from the (-)-ends and an increase of the free monomer

concentration (60). Capping proteins are also present in cells to cap filament ends, inhibiting

elongation (61). These proteins regulate the dynamics of the actin network by controlling the

length of filaments, preventing an excess of filaments, and ensuring G-actin concentrations are

sufficient for various cellular activities.

1.2 Experimental Methods for Actin Polymerization on Modified Substrata

Several previous experimental methods have been used to measure the extent of F-actin

elongation and calculate the elongation rate in vitro. These include capillary viscometry,

difference spectra, and fluorescence with pyrene-labeled actin monomers (62). The major

drawbacks of these methods are that filament lengths can not always be determined, and

elongation can be monitored, but not visualized, in real-time. Since single actin filaments are

7 nm in diameter, the only ways to observe these filaments are through fluorescent labeling or

electron microscopy (63, 64). Previous experiments used phalloidin, a toxin that binds to and

fixes actin filaments to prevent them from depolymerizing, in order to measure the length and

structure of actin filaments with fluorescent microscopy (63, 65-67). Recently, polymerization

assays have been enhanced by total internal reflection fluorescence (TIRF) microscopy, which

allows for real-time visualization of elongating actin filaments bound to or near a surface.

Manipulation of the substrata through microcontact printing, a technique used for a wide variety

of applications, including confinement of cell growth and proteins to a specific pattern, can

provide spatial control over filament binding to surfaces.

1.2.1 Total Internal Reflection Fluorescence Microscopy

Until recently, fluorescently-labeled phalloidin was the best method for visualization of

filaments in fluorescence because fluorescently-labeling actin monomers directly caused a high

background signal from the solution, flooding out the resolution of filaments. Because both EM









and phalloidin-labeling fix the filaments at a static point, these techniques still prevent

visualization of filament elongation in real-time. The development of TIRF microscopy allows

for filaments bound to surfaces to be visualized in a solution of fluorescently-labeled monomers

by only illuminating the 200 nm near the surface of the sample, thus minimizing background

fluorescence. Therefore, any fluorophores within the bulk of the sample are not excited, and

signals from the excited fluorophores that do emit light are not obscured by any out-of-focus

fluorophores (68).

The basis of TIRF microscopy is the evanescent wave created when the light is totally

internally reflected at an interface between two mediums with different refractive indices.

Specifically, the light travels from a medium with a high refractive index to one with a low

refractive index. The evanescent wave is created when light is reflected at an angle equal to or

lower than the critical angle, 09, and has the same frequency as the incident light but propagates

parallel to the interface (Figure 1-2). The critical angle is calculated from Snell's law (Equation

1-1) and is dependent on the refractive indices of the two mediums at the interface.

ni sin 01 = n2 sin 02 (1-1)

The refractive index of the aqueous buffer phase is n2, and nl is the refractive index of the glass

phase. At the critical angle, refraction occurs at an angle of 900; therefore sin 02 = 1, and the

critical angle is defined by Equation 1-2.

sin 0, = n2/nl < 1 (1-2)

Typically, a laser beam travels through a glass medium first (n = 1.52). Contacting this

layer is an aqueous buffer phase containing the specimen to be observed. This solution phase

has a lower refractive index (n = 1.33-1.37 for typical aqueous mediums) than the solid phase.









The intensity of the evanescent wave decreases exponentially from the surface to approximately

200 nm, at which point it disappears (Equation 1-3).

I(z) = I(0)e (1-3)

The intensity at any position, I(z) is exponentially decreased from the initial intensity I(0) by a

function of the ratio of the position from the interface and the penetration depth, d (Equation

1-4).

d =o(n2sin2 n 2)1 (1-4)
47c

The penetration depth is dependent on the refractive indices, the angle of incidence, and the

wavelength of the incident light in a vacuum, Xo (68).

Actin filaments containing fluorescently-labeled monomers bound to a surface are clearly

resolved in TIRF. One way to attach filaments is by adsorbing N-ethylmaleimide

(NEM)-myosin to glass (69, 70). NEM binds to thiol groups on myosin, irreversibly inactivating

the catalytic site for ATP hydrolysis (71, 72). This allows an actin filament to remain attached to

the myosin molecule (Figure 1-2). Amann and Pollard directly observed individual filaments

formed from a solution of G-actin partially labeled with rhodamine by using NEM-myosin to

bind the filaments to the surface. Their experiments confirmed the elongation rate constants of

the (+)-ends of actin filaments as previously found by EM. They also determined that

rhodamine-labeled actin was a kinetically inactive label, making it useful for visualizing

elongating filaments (73). They showed that Arp2/3 branching in solution occurred at random

sites along the filament length, confirming previously published results that used phalloidin

stabilization (73). Kuhn and Pollard studied both polymerization and depolymerization rates









further using TIRF to visualize filament (+)- and (-)-end elongation in real-time under a variety

of conditions (70).

1.2.2 Microcontact Printing

In our experiments, we patterned NEM-myosin on glass coverslips using microcontact

printing to bind actin filaments to the surface and guide their elongation (Figure 1-3).

Microcontact printing is part of a group of techniques known as soft lithography, which uses a

patterned elastomer as a stamp, mold, or mask in replacement of a rigid photomask associated

with photolithography to create micropattems (74). Polydimethylsiloxane (PDMS) elastomers

are most commonly used for microcontact printing because of a number of advantages, including

chemical and thermal stability and durability. This technique was initially used to form

self-assembled monolayers (SAMS), or monolayers that form due to the spontaneous

aggregation and organization of molecules into a stable structure without the use of covalent

bonds (74). Kumar and Whitesides first demonstrated this technique by inking patterned PDMS

with feature sizes 1 to 100 .im with hexadecanethiol and then stamping it onto a gold surface

(75). The applications of microcontact-printed SAMS were furthered by the selective deposition

of other materials onto the monolayers, creating the same pattern with a different exposed

material. These materials included conducting polymers, inorganic salts, metals, and proteins

(74).

Microcontact printing has more recently expanded to transfer metals, electromagnetic

particles, electrochemical particles, and proteins directly. Kind, et al. demonstrated the inking of

a solution of Palladium(II) onto a Titanium-coated silicon wafer (76). Iron dots patterned onto a

magnetic slave-film have successfully duplicated magnetic signals (77, 78). Applications have

also moved toward biology, with Renault, et al. showing that microcontact printing

fluorescently-labeled antibodies could provide a method for studying single protein molecules on









surfaces (79). Xu, et al. used a PDMS stamp to transfer patterns ofE. coli onto an agarose

substrate (80). Furthermore, microcontact printing collagen-like proteins has been shown to

restrict cell growth to specified regions of surfaces (81).

Microcontact printing can be used for a broad span of applications, however, there are still

problems associated with this technique. Distorted patterns may occur due to structural

deformation during contact of the surfaces and are enhanced as the dimensions of the stamp

become submicron (82). These deformations are inherent due to the elastomeric nature of the

polymer. Additionally, an excess of ink and the diffusion of non-covalently bound molecules to

unprinted regions of the surface may cause dimensions of the printed pattern to become larger

than those on the stamp (82).

1.3 Actin-Based Motility

Actin-based motility occurs when actin filaments elongate against the surface of a motile

object, propelling the object forward to make space for the addition of new monomers. Listeria,

a classic example of actin-based motility, forms an actin rocket tail consisting of thousands of

filaments cross-linked into a bundle. Filaments in the tail elongate near the surface of the

bacteria, pushing against the bacteria and propelling it forward. Many proteins have been shown

to be involved in various types of actin-based motility, including the Listeria transmembrane

protein ActA and vasodilator-stimulated phosphoprotein (VASP), which may act together as an

end-tracking motor (25, 26). These and another group of proteins (formins) have been used in

vitro to replicate actin-based motility with bacteria and various types of particles.

1.3.1 Listeria and Particle Motility

Motility of bacteria in cells, such as Listeria, occurs through the recruitment of actin

monomers from the host cell's cytoskeleton and the subsequent generation of force through the

formation and polymerization of many bundled, cross-linked actin filaments actinn rocket tail) at









the rear of the bacterium (28, 29). Listeria motility has been successfully reconstituted in vitro in

various cell extracts and conditions (83, 84). Although the exact mechanism is unclear (25, 26,

85, 86), it is well-known that the surface protein ActA bound to the membrane of motile Listeria

is a key protein required for the actin rocket tail formation (87). Current experimental results

support the role of ActA and VASP as an end-tracking motor, providing persistent attachment

between the filaments and the motile object.

By extracting ActA protein from Listeria, polystyrene beads can be functionalized with

ActA to mimic Listeria motility in vitro (88, 89). Beads provide the opportunity to study the

mechanism and optimize motility by controlling important parameters, such as diameter and

protein surface density of the motile object. Cameron, et al. (1999) successfully used an

extraction and purification of histadine-tagged ActA from Listeria that was incubated on

carboxylated polystyrene beads to coat their surfaces to replicate actin-based motility systems.

They found that particle diameter affected the fraction of particles with tails and the velocity of

the particles (90).

Experimentally, Cameron, et al. (2001) discovered a single actin filament attached to a

50-nm bead with 37.5% surface coverage of ActA. No average velocity was reported due to

thermal fluctuations of the bead and limitations of the imaging equipment (89). However, EM

images showed that no 50-nm bead had more than one actin filament attached to it. The length

of the actin filament attached to the 50-nm bead indicates that the (+)-end of the filament must

have been attached to the bead persistently during elongation, otherwise the bead would have

diffused away from the actin filament during fluctuations from the surface for monomer addition

(25).









Other studies have supported end-tracking motors as the mechanism for ActA/VASP

mediated actin-based motility while also demonstrating the ability of the ActA/VASP complex to

support in vitro actin-based motility of bacteria and vesicles. Kuo and McGrath were able to

track the trailing ends of Listeria using an optical trapping and laser particle tracking system.

They showed that Listeria moves with 5.4 nm steps with pauses in between each step. During

the pauses, the bacteria fluctuated 1.31 0.004 nm parallel to and 0.94 0.03 nm perpendicular

to the stepwise movement. This was 20-fold less fluctuation than seen by lipid droplets placed

next to the bacterium in the same solution, indicating that the actin tail must be coupled to the

motile surface (91).

Lipid vesicles undergoing deformation and motility due to actin polymerization was first

induced by Upadhyaya, et al. (92). By incubating fluorescently-labeled ActA on the surface of

the vesicles, ActA polarized to the end of the vesicle where the actin tail formed. Data showing

the vesicle trailing end velocity to be 2.5 [tm/min (six times faster than the vesicles initial

velocity) at a "snapping point", caused by a release of the vesicle, led to the conclusion that the

actin was persistently attached to the ActA on the motile surface (92).

Formins, multi-domain proteins that have been found to regulate polymerization of

unbranched actin filaments, have also been used to reconstitute actin polymerization in vitro.

The FH1 domain of formins binds profilin (93) while the FH2 domain binds the (+)-end of the

actin filament (27, 94-96). Romero, et al. adsorbed the FH1-FH2 domain from the formin

mDial onto polystyrene beads and demonstrated a tight coupling between the polymerization

and ATP hydrolysis on filaments attached to FH1-FH2 (27). In vitro, both ActA- and

formin-induced motility provide consistent results for actin-based motility of particles, bacteria,

and vesicles, all of which can be exploited in bionanodevices.









1.3.2 Mechanisms

The proteins ActA and VASP are responsible for actin-based motility of Listeria, but

currently, the mechanism as to how they produce forces is under debate. In the Brownian

Ratchet model (97, 98), force is generated by actin polymerization of free-ended filaments

impinging on a surface or object. The free energy of ATP-actin monomer addition to the

filament (+)-ends is converted into the driving force for the motility (99). The actin filaments are

assumed not to be attached to the surface but are stabilized in the cytoplasm through branching

and cross-linking.

Thermal fluctuations of the filament (+)-end away from the surface allow the time and

space needed for a monomer to add onto the end of the filament. Because the filament cannot

diffuse backwards due to the cross-linked network, it pushes forward against the motile surface

as it elongates. Based on thermodynamics, the work done on the motile surface can not exceed

the free energy released upon monomer addition, given by Equation 1-5.

[AT, ]
AG(+)add k -kT[n A (1-5)
[A ] (+)crlt


The Boltzmann constant and the absolute temperature, kT, represents the thermal energy,

[AT](+)crit is the (+)-end critical concentration of ATP-actin, and [AT] is the total concentration of

G-actin. When depolymerization at the (-)-end of the filament is occurring, the concentration of

actin monomers must be less then the critical concentration at the (-)-end. Based on this

concentration estimation and the length per monomer addition of 2.7 nm, the maximum force

that a filament can apply to a surface by monomer addition alone is 2 pN. This theoretical force

is smaller than previously published force estimations used for steady-state elongation (86, 100).

In the actoclampin model, elongating filaments are persistently attached to the surface

through a multivalent surface-bound protein that interacts with high affinity for ATP-actin









monomers. The free energy released upon hydrolysis of ATP is assumed to drive affinity

modulations between the end-tracking proteins and the filament (+)-end in a way that converts

the hydrolysis energy into the mechanical work used for inserting new monomers and pushing on

the motile surface. End-tracking motors may also provide some protection of elongating filament

ends from capping proteins while transferring a profilin-actin complex into the filament (26). In

addition to the 2 kT of energy available from the addition of one subunit, actoclampin can utilize

as much as 14 kT per subunit of free energy from ATP hydrolysis (25).

Experimental results, including the dependence on profilin and ATP hydrolysis, show that

formins behave like "actoclampin" filament end-tracking motors (25-27, 101, 102). In the case

of Listeria, the putative actoclampin motor includes both VASP and ActA (26). VASP contains

an EVH2 domain, which binds both G-actin and F-actin, and an EVH1 domain, which has

binding sites on ActA. VASP also contains a proline-rich domain which is thought to recruit

profilin-actin for subsequent addition onto the filament (+)-ends (103). Supporting this proposed

role of profilin in actin-based motility by actoclampin, fluorescently-labeled profilin has been

observed to localize to Listeria's surface during motility, with the speed of Listeria directly

proportional to the fluorescent intensity (104).

1.4 Bionanodevices

Both actomyosin and a similar system using kinesin with associated microtubules have

been exploited to transport material in vitro. Two different methods exist for the mechanism of

transport. The first is the sliding filament assay which relies on immobilized myosin or kinesin

to transport actin filaments or microtubules, respectively. The second assay is the immobilized

filament assay, which binds actin filaments or microtubules to a surface to create a path for

myosin or kinesin to walk along. Both have been shown to transport cargo in a directive manner.









1.4.1 Sliding Filament Assay

The sliding filament assay is more commonly used than the immobilized filament assay

because it is easier to set up and execute experiments (32). Typically, surfaces contain a region

with high affinity for protein adsorption and a region with low affinity. Bunk, et al. found that

the photoresist materials MRL-6000.1XP and ZEP-520 both efficiently adsorbed heavy

meromyosin (HMM) and subsequently bound and moved actin filaments in contrast to

PMMA-200, PMMA-950, and MRI-9030, which exhibited poor motility. For this reason, they

created 100 to 200 nm grooves in a material layer exhibiting low motility (PMMA-950) to

expose an underneath layer with high motility behavior (MRL-6000.1XP) to efficiently guide

actin filaments (8). Another method for surface modification includes the use of high-resolution

e-beam patterning exposure of a poly[(tert-butyl-methacrylate)-co-(methyl methyl methacrylate)]

to make hydrophobic (high-energy exposure) and hydrophilic (low-energy exposure) regions, the

latter with a low affinity for HMM adsorption and subsequent filament binding (19).

Microcontact printing was used to create SAMs on gold surfaces and attach biotinylated myosin

to the patterned region. This method ensured the orientation of the myosin would be optimal for

gliding actin filaments (18).

Clemmens, et al.(2003) tested kinesin adsorption and the guidance of microtubule motility

on three different designs: a chemical border between ppPEO (low affinity for kinesin) and glass,

a channel with floors and walls made from polyurethane, and a combination of the two, using

SU8-PEO photoresist to make non-adhesive walls with channel floors made of SiO2 for

adsorption of kinesin. The most efficient design was the latter, which combined both chemical

definition and topographical boundaries (105). This design was further optimized by using

AZ5214 as the non-adsorbing photoresist on glass and creating channel walls with an undercut to

prevent microtubules from climbing over the sides of the channels (17).









Sliding assays have also allowed for the exploration of many different track shapes, which

may be beneficial for sorting the direction of the moving filaments. Suzuki, et al. observed actin

filaments moving in concentric circles, letters, and figure eights on PMMA surfaces (21). Paths

of microtubules have been studied in channels using different crossing junctions, including

tangent circles and crosses, as well as concentric circles (16). Additionally, the sorting of

microtubules has been examined by using arrow heads or tangent, straight channels shooting off

of circular paths (12, 13, 16). These types of devices are designed to catch any microtubule

traveling in an opposite direction in these "traps" and allow for the filament to turn around. The

ultimate goal is for all filaments to be moving in the same direction.

1.4.2 Immobilized Filament Assay

Fewer attempts have been made to immobilize filaments for myosin- or kinesin-mediated

cargo transport. Glass particles attached to kinesin have successfully traveled along

gluteraldehyde-immobilized, isopolar microtubule arrays (3). Nanocrystals have also been

shown to travel along microtubule arrays fixed to the surface by poly-L-lysine (5). Reuther,

et al. has biotemplated microtubules into single-molecule tracks, which bind kinesin. These

immobilized filaments successfully guided another microtubule along the path by binding

kinesin to the immobilized microtubules (22). Actin paracrystal structures formed in lipid

bilayers have been shown to guide myosin-coated glass beads (6). Immobilized-filament assays

have the potential to guide myosin (or kinesin)-mediated cargo unidirectionally by orienting the

filament (+)-ends to point in the same direction (32). One attempt to align filament polarity

successfully used electrostatic condensation of F-actin/gelsolin to create an unidirectional track

for myosin-bound bead transport (19).









1.4.3 Material Transport

An important aspect of all of these techniques is that they are able to transport material in a

controlled manner. In bionanotechnology, particles have the advantage of being functionalized

to not only move by actin-based motility but also to bind to specific proteins or molecules that

need to be transported against flow or diffusion gradients. As mentioned above, guidance of

various particles bound to kinesin and myosin have been attempted. Quantum dots (7.6 nm

diameter) functionalized with kinesin were observed moving along microtubule tracks at an

average velocity of 0.28 rim/sec, for distances ranging from 1 to 5 rim, with the number of

events falling exponentially as the distance increased (5). Larger kinesin-coated spherical

particles (1.8 to 2.8 itm) made from glass, polystyrene, and paramagnetic polystyrene traveled

along isopolar microtubules for 1 to 2.5 mm at a rate of approximately 0.5 itm/sec. Suda and

Ishikawa found that myosin-bound particles with 65 [tm diameters slide at rates of 338 itm/sec

on actin filament paracrystal structures (6).

Sliding actin filaments and microtubules have also been shown to carry attached cargo.

Bachand, et al. showed the transport of quantum dots attached to microtubules along a

kinesin-coated surface (2). Similarly, Mansson, et al. showed transport of quantum dots attached

to actin filaments on a myosin-coated surface (4). This same technique has been used for motile

microtubules labeled with antibody sandwiches, i.e. the microtubule can selectively bind a target

antigen and then a secondary antibody for detection (14). Suzuki, et al. successfully transported

a gelsolin-coated polystyrene bead (1 [im diameter) attached to the (+)-end of an actin filament

on a myosin surface (7). All of these examples demonstrate the ability to successfully transport

cargo against flow and diffusion gradients through a variety of mechanisms, all using

biomolecular motors.













*2SZ% Rt~ (-)-end


ATP

A----

AP


SATP-actin

ADP-actin

ADP-Pi-actin


Figure 1-1. Treadmilling of an actin filament. ATP-actin binds to the (+)-end of the filament.
The ATP hydrolyzes, forming an intermediate monomer in the filament,
ADP-Pi-actin. The phosphate is eventually released, leaving the remaining monomers
in the filament as ADP-actin. The monomers at the (-)-end dissociate from the
filament and are released back into the solution, where the ADP molecule is
exchanged for an ATP molecule, and the cycle repeats.


Single Actin
Filament



TV-IMyosuhi --


Figure 1-2. Experimental set-up for binding filaments to a surface. NEM-myosin is adsorbed to
a glass coverslip, where it binds a polymerizing actin filament. A laser beam is
totally internally reflected at the interface of the glass coverslip and the sample
solution. This creates the electromagnetic (EM) wave, known as the evanescent
wave, which excites any fluorescently-labeled molecules attached to monomers that
have incorporated into the actin filament.


(+)-end


Pi













Adsorption
of Pro rein
Solution


Patterned PDMS



S e
IM


Invert and
Transfer


Panre ued
Substrata


"-M r- I r-


Figure 1-3. Schematic of microcontact printing protein on glass. A patterned piece of PDMS is
incubated with a solution of protein. The stamp is dried and placed in contact with
the glass surface. When the stamp is removed from the glass, the protein that was
directly contacting the glass is transferred onto the glass in the corresponding pattern.


7 -









CHAPTER 2
GUIDANCE OF ACTIN FILAMENT ELONGATION ON FILAMENT-BINDING TRACKS

2.1 Introduction

Guidance of single actin filament elongation is a simplified system to study the potential of

actin-based motility for applications in nanotechnology. By directing single actin filament

elongation, controlled tracks of actin filaments for cargo-carrying myosin can be created. In

addition, single-filament propulsion of small particles (-50 nm in diameter) is predicted to

behave in a similar way on filament-binding tracks as elongating actin filaments without

particles. We developed and characterized the ability of microcontact-printed tracks of

immobilized filament-binding proteins (NEM-modified myosin) to guide the direction of

filament elongation. The direction and rate of polymerization of individual surface-bound

filaments were directly observed and quantified using TIRF microscopy (69, 70). Printed

NEM-myosin tracks were found to capture nascent filaments from solution and guide their

subsequent elongation to a degree that was dependent on the track width and myosin density.

These findings suggest a mechanism whereby the myosin track can recapture the tips of

undulating filaments that cross track boundaries at sufficiently small glancing angles.

2.2 Materials and Methods

2.2.1 Protein Preparation

Actin was polymerized from a mixture of unlabeled and fluorescently-labeled G-actin. To

prepare unlabeled actin, we resuspended commercial G-actin that was lyophilized as a 10 mg/mL

actin solution in 5 mM Tris-HCl pH 8.0, 0.2 mM CaC12, 0.2 mM ATP, 5% sucrose and 1%

dextran (Cytoskeleton, Inc., Denver, CO) in 25 tL deionized H20 and incubated the sample on

ice for at least 2 h to dissolve the protein thoroughly. After 24-h dialysis against G-buffer (5 mM

Tris-HCl pH 8.0, 0.01% NaN3, 0.1 mM CaC12, 0.5 mM Dithiothreitol (DTT), 0.2 mM ATP),









with three buffer exchanges, actin was collected and clarified by centrifugation at 107,000 x g

for 2 h at 4 C in a TLA100.2 rotor (Beckman Coulter, Inc., Fullerton, CA). G-actin was stable

at 4 C for up to two weeks. Lyophilized actin with random surface lysine residues covalently

bound to an activated ester of rhodamine (Cytoskeleton, Inc., Denver, CO) was used (without

dialysis) at a stock solution concentration of 23 tM after the powder was resuspended in

deionized water for three days at 4 OC.

To prepare Alexa 488-labeled actin (73), purified rabbit muscle G-actin (106) was

polymerized at a concentration of 60 tM by dialysis against polymerization buffer (2 mM

3-morpholinopropanesulfonic acid (MOPS) pH 7.5, 50 mM KC1, 1 mM MgC12, 0.2 mM ATP).

A 5-fold molar excess (300 gM) of Alexa Fluor 488 carboxylic acid, succinimidyl ester

(Invitrogen, Molecular Probes, Carlsbad, CA) in dimethyl sulfoxide (DMSO) was added to the

polymerized actin and left overnight at 4 OC. Labeled F-actin was then centrifuged at

288,000 x g for 30 min at 4 OC in the TLA100.2 rotor. G-actin was prepared from this pellet by

resuspension and 48-h dialysis against G-buffer. Monomeric actin was collected and centrifuged

at 107,000 x g for 2 h at 4 C to remove oligomers. The monomer-rich supernatant was

collected, aliquoted, rapidly frozen in liquid nitrogen, and stored in -70 C for up to six months.

For actin filament binding in the absence of myosin-mediated motility, myosin II ATPase

activity was inactivated by N-ethylmaleimide (NEM) treatment, as described elsewhere (70).

The contents of five tubes of commercial lyophilized 95%-pure myosin II (Cytoskeleton, Inc.

Denver, CO) was resuspended in 250 |L deionized H20, yielding a 20 mg/mL myosin solution.

After 2-h dialysis at 4 OC against 10 mM Imidazole, pH 7.0, 10 mM ethylene diamine tetraacetic

acid (EDTA), and 500 mM KC1 myosinn dialysis buffer), the protein was diluted to 10 tM in the

same buffer, and reacted with 1 mM of NEM for 1 h on ice. DTT (1 mM) was used to quench









the unreacted NEM for an additional 1 h on ice. After overnight dialysis at 4 OC against 10 mM

Imidazole, pH 7.0, 500 mM KC1, 10 mM EDTA, and 1 mM DTT, with one additional buffer

exchange, NEM-myosin was aliquoted, rapidly frozen in liquid nitrogen, and stored at -70 OC for

up to six months.

Purified, precipitated profilin expressed from Escherichia coli and stored in lysis buffer

(20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT, and 1 mM

phenylmethanesulphonyl-fluoride (PMSF)) in the presence of 40 mM NaCl was generously

donated by Dr. William Zeile. Profilin precipitate was spun at 16,000 x g for 20 min in an

Eppendorf 5415C microcentrifuge, resuspended in 4x polymerization buffer (20 mM Imidazole

pH 7.0, 200 mM KC1, 4 mM MgC12, and 4 mM ethylene glycol tetraacetic acid (EGTA)), and

dialyzed for 48 h against the same buffer at 4 OC.

2.2.2 Microcontact Printing

NEM-myosin was microcontact-printed as parallel tracks on glass coverslips with the aid

of a polydimethylsiloxane (PDMS) stamp. A silicon wafer mold of desired pattern was created

by light exposure of SU8 photoresist (Microchem Corporation, Newton, MA) using a Karl Stiss

MA-6 photoresist microlithographer. A mixture of 10:1 prepolymer to catalyst of Sylgard 184

(Dow Coming Corporation, Midland, MI) was degassed and poured onto the surface-fluorinated

silicon wafer, after which PDMS was polymerized in the mold at 60 OC for 1 h (107). The

PDMS stamps were generously made and donated by Dr. Suzanne Ciftan-Hens (International

Technology Center, Raleigh, NC).

The resulting hydrophobic PDMS stamps (with tracks ranging from 3 20 [tm wide) were

inked for 40 min with a NEM-myosin solution, washed three times with myosin dialysis buffer,

three times with deionized H20, and dried under nitrogen (79, 107). After acid-washing and

treatment as described elsewhere (70), glass coverslips (No. 1, Fisher Scientific, Waltham, MA)









were pressed and left in contact with a stamp's inked-surface for 20 min. The resulting stamped

coverslips were either examined by atomic force microscopy (AFM Dimension 3100, Veeco,

Woodbury, NY) or used in hand-fabricated flow-cells constructed by adhering the inverted

coverslip onto a microscope slide with double-sided Scotch tape. Until use, flow-cells were

stored in myosin dialysis buffer for up to 6 h.

2.2.3 Actin Polymerization

Actin filaments were polymerized according to a previously published protocol (70).

Actin monomer solution was combined with a 10x magnesium-exchange buffer (10 mM EDTA,

1 mM MgCl2) and incubated on ice for 5 min to convert Ca-ATP-actin to Mg-ATP-actin. The

Mg-ATP-actin was then mixed with a 2x concentrated polymerization buffer (20 mM Imidazole

pH 7.0, 100 mM KC1, 2 mM MgC12, 2 mM Ethyleneglycol-bis(P-aminoethyl)-

N,N,N',N'-tetraacetic Acid (EGTA), 20 mM DTT, 0.4 mM ATP, 1% methylcellulose, 30 mM

glucose, 40 [tg/mL catalase and 200 [tg/mL glucose oxidase) for a final concentration of 0.75 to

1.5 [aM actin (15-30% fluorescently-labeled). This solution was immediately transferred into the

flow-cell.

Prior to experiments, flow-cells containing cleaned glass coverslips were treated according

to Kuhn and Pollard (70), with 0.2 gM NEM-myosin for 1 min followed by 1% bovine serum

albumin (BSA) in 50 mM Tris-HC1, pH 7.6, 600 mM NaCl (HS-TBS) to remove any unbound

NEM-myosin, followed by washing with 1% BSA in 50 mM Tris-HC1, pH 7.6, 50 mM NaCl

(LS-TBS) to remove any unbound NEM-myosin, lower the salt concentration in the flow

chamber, and to passivate any exposed glass (70). The volume of each wash was approximately

twice the chamber volume. Flow-cells possessing NEM-myosin-stamped coverslips were washed

with the 1% BSA in 50 mM Tris-HC1, pH 7.6, 600 mM NaCl and 1% BSA in 50 mM Tris-HC1,

pH 7.6, 50 mM NaCl before actin solution was added.









2.2.4 TIRF Microscopy and Data Analysis

To observe our samples, we used a commercial objective-based TIRF consisting of a

Nikon Eclipse TE1200 inverted microscope fitted with TIRF optics (Nikon Instruments,

Melville, NY) and an Argon 488-nm or a Helium-Neon 532-nm laser (Melles Griot, Carlsbad,

CA). Images were acquired using a digital charge-coupled device (CCD) camera (QImaging,

Burnaby, BC, Canada) and analyzed using MetaMorph software (Molecular Devices

Corporation, Sunnyvale, CA). Time-lapse images of each field-of-view were taken at 5-sec

intervals.

To quantify filament orientation, the filament images were first discretized into segments

by selecting points spaced 0.1 3 atm apart along the filament length. We only analyzed those

filaments that were elongating during the observation time. Filaments that spanned the

NEM-myosin track border were discretized up until the track edge. Furthermore, to eliminate

the possibility that interacting filaments might bias the data, images containing a high density of

elongating and non-elongating filaments near the surface were excluded. The minimum number

of independent tracks for each track width was ten, while each track width contained

measurements from at least 84 filaments. The minimum number of independent tracks for one

NEM-myosin treatment concentration was ten, while each condition contained measurements

from at least 86 filaments.

Taking 0 as the angle between the segment and the track direction, the mean of cos(20)

(weighted by segment lengths) for each filament was calculated. A mean of cos(20) equal to one

corresponds to perfect alignment, and a value of zero indicates random alignment. An overall

cos(20) for each track width was calculated by averaging the cos(20) values of the filaments,

weighted by the filament length. Data were collected from samples run on different days, but a

one-way ANOVA between the samples for each track width concluded that no statistical









differences were present between days. Therefore, each individual track was considered an

independent sample and was used to determine the standard error for each condition.

The weighted mean cos(20) was plotted against both track widths and NEM-myosin

treatment concentrations with error bars representing the standard error (SE) based on a

weighted standard deviation (SD). To determine if the alignment of the filaments was

significant, a one-sample t-test was used to calculate a one-tailed p-value by comparing the

average cos(20) for each track width and NEM-myosin concentration with a cos(20) value of

zero for random alignment. The highest and lowest NEM-myosin treatment concentrations were

compared using a two-sample t-test. Filaments within 1.5 tm of the track edge were pooled to

obtain alignment near the edge of the track. In addition, the average distance of each filament

segment from the edge of the track was estimated based on positions obtained manually from

MetaMorph, and the distances were plotted against the alignment for each segment to create

scatter plots for all samples.

Changes in filament length were measured for a minimum of three time-points per

filament, allowing us to calculate the elongation rate of filaments for each NEM-myosin and

profilin concentration from a line-fit of filament length versus time (with each 0-time intercept

set to 0). Instantaneous elongation rates, with subunits/sec values (based on 370 subunits per [tm

filament length), were pooled from at least 12 filaments for each NEM-myosin concentration and

at least 13 filaments for each profilin concentration. Elongation was observed and measured at

filament (+)-ends only. Elongation rates for the four NEM-myosin treatment concentrations

were tested for statistical differences using a one-way ANOVA.









2.3 Results

2.3.1 Filament Binding to Stamped Surfaces

Using the microcontact-printing procedure, we produced and characterized filament

growth on myosin tracks of six different average widths SD (3.3 0.3, 4.3 0.5, 5.7 0.2,

10.7 1, 15.1 0.8, 20.2 0.5 [tm). Fluorescence and AFM images were taken to assess the

uniformity of NEM-myosin coverage. As shown in Figure 2-1A, the NEM-myosin regions were

clearly delineated by the preferential non-specific adsorption of fluorescent actin monomers or

oligomers with the NEM-myosin regions relative to the BSA regions. The magnified

three-dimensional AFM image (Figure 2-1B) of a representative track edge shows a confluent

protein layer, with thickness shown in Figure 2-1C. The measured thickness variation of the

protein layer is comparable to the 7-nm diameter of a single actin filament, implying that the

peaks and valleys of the layer should have little effect on the guidance of the filaments.

The microcontact-printed surfaces were exposed to the actin solution, allowing capture of

nascent filaments from solution by the NEM-myosin-coated regions with the majority of

captured filaments continuing to elongate on the surface. (Surfaces stamped with 0.5 mg/mL

BSA showed little evidence of filament binding, as shown in Figure 2-2.) We were able to

monitor (+)-end filament elongation on the NEM-myosin tracks in real-time using TIRF

microscopy (Figure 2-3). Notably, the tips of myosin-bound filaments clearly undulate during

elongation, and the filaments continue to bind to NEM-myosin along their length as they extend.

While filament segments were found both on the tracks and on the BSA-coated regions, filament

segments on the BSA regions underwent larger thermal undulations (observed in the x- and

y-direction) than those confined on the NEM-myosin track, indicating that filaments were tightly

bound only to the NEM-myosin tracks (Figure 2-4). Filaments initially bound to the track either

remained in the NEM-myosin region or elongated beyond the track edge, with the filament end









continuing to elongate and undulate over the BSA-coated region (Figure 2-5). In many cases,

these undulations allowed a filament which had crossed the boundary of the NEM-myosin track

at a glancing angle to rebind to the track, now with the filament end often aligned with the track

edge (Figure 2-6).

2.3.2 Alignment of Filaments

To determine the effect of the NEM-myosin tracks on the filament alignment, we first

eliminated samples containing a high density of actin filaments on the surface in the track region

(Figure 2-7). The high density of filaments near the surface may increase filament-filament

interactions, which could then increase the alignment of the filaments with each other. Also, the

dense samples were difficult to analyze because individual filaments were not always

distinguishable.

The resulting alignment of the filaments with the track edge was quantified by measuring

the angle 0 for segments along each of several filaments for each track width (Figure 2-8A) and

calculating the mean of cos(20). Filament alignment on the narrower track widths was found to

be significant and the degree of alignment increased as the track width decreased (Figure 2-8B,

solid squares). To determine whether alignment resulted primarily from interactions with track

boundaries, the mean of cos(20) was re-calculated for the subset of filaments within

approximately 1.5 [tm of the track edge on tracks wider than 3.3 |tm (Figure 2-8B, open

squares). The filaments near the track boundaries had a similar degree of alignment independent

of the track width. These observed trends are consistent with the interpretation that filaments in

tracks with widths comparable to the filament persistence length (- 10 [tm (48, 49)) take random

walk trajectories within the boundaries of the track and only become aligned when encountering

the track boundary. On the other hand, filaments in tracks that are small relative to the filament









persistence length encounter the track boundary and become realigned before significant changes

in direction occur (Figure 2-8C).

The probability of realignment of a filament with the track edge is also dependent on the

alignment of the filament immediately before crossing the NEM-myosin track edge. The

histogram in Figure 2-9 demonstrates that at smaller alignment angles, these rebinding events

were more frequent, ranging from -90% recapture-probability for angles less than 150

decreasing to zero probability at angles greater than 600. From this result, we surmise that the

bending energy at large angles required to recapture a filament is too large to be achieved by

thermal undulations. To confirm that thermal undulations caused this bending, we estimated the

bending energy of several filaments from Equation 2-1 (108).


Ebend = kBT -d'ds (2-1)
2 Jds

The thermal energy is kBT (Boltzmann constant multiplied by temperature), A is the filament

persistence length (- 10 [tm (48, 49)), and s is the arc length of a filament that crossed the

boundary and then bent back to be captured by the track. In each case, Ebendwas on the order of

kBT, suggesting that changes in filament direction were driven by thermal energy (49).

A scatter plot shows all of the filament segments and their alignment as a function of

distance from the edge of the track (Figure 2-10). Segments seem to be concentrated near the

edge at both aligned (cos(20) = 1) and unaligned angles (cos(20) = -1). This suggests when

filaments reach the edge of the track, they tend to realign at small angles, increasing alignment,

or they leave the track completely at large angles, making the distribution of segment angles near

the track edges at the extremities.









2.3.3 NEM-Myosin Concentration

We also investigated the effect of NEM-myosin concentration used to treat the PDMS

stamps on the alignment of elongating filaments. As shown in Figure 2-11A, the degree of

alignment, again quantified by the mean cos(20), trended upward with NEM-myosin treatment

concentration, with the degree of alignment on tracks prepared with the highest concentration

tested (2 pM) significantly greater than that of the lowest (0.1 [M), the latter of which failed to

generate statistically significant alignment. These results suggest that lower NEM-myosin

surface densities are less efficient at recapturing undulating filament ends that have elongated off

the track edge. Finally, we anticipated that at higher densities, myosin might bind at or near the

filament (+)-ends and thereby hinder monomer incorporation. To determine the effect of myosin

concentration, we measured the elongation rate of filaments on the protein tracks prepared with

varying NEM-myosin concentrations used in Figure 2-11A. As shown in Figure 2-11B, the

elongation rate decreased only slightly for the highest myosin concentration, implying that the

NEM-myosin had little effect on actin polymerization.

Scatter plots of segment distance from the track edge versus filament alignment for each

NEM-myosin concentration are shown in Figure 2-12. The 0.1 kM NEM-myosin treated sample

has the least bias of filament segments aligned near the edge of the track, presumably due to the

low concentration of myosin available to rebind and realign filaments. The 0.5 and 2 gM

samples show a slight bias toward aligned filament segments. The 1 PM sample, which had the

most segments available for analysis, is the subset of the filaments in Figure 2-10 that were on a

5 pm wide track. Therefore, the 1 kM sample shows the same trend as Figure 2-10, again

indicating that the alignment of filament segments closer to the track edges is independent of

track width.









2.3.4 Control of Actin Polymerization

Figure 2-13 shows a sample in which filaments accumulated within the BSA regions

(spaces where NEM-myosin was not stamped). Although these filaments undergo large thermal

undulations, unlike those that are bound onto the NEM-myosin tracks, their accumulation seems

to prevent filaments from binding to the NEM-myosin track. These interactions could be due to

NEM-myosin molecules that adsorbed onto an area that was not within the stamp, interactions

between actin filaments, nonspecific interactions between actin filaments and BSA or glass, or a

combination of all three effects. The density of filaments in these regions is observed to increase

over time, presumably because of the strong effects actin filaments have on each other. In

addition, if the filaments are diffusing to the surface as long filaments (lengths greater than the

track width), the filaments at most may lay across the track at some angle rather than align with

the track.

One way to potentially prevent this behavior is to control actin polymerization through

nucleation to prevent filaments from accumulating near the surface within a short time frame.

Protein samples may contain small oligomers that cause the density of filaments to increase at

much higher rates than other samples. Also, the lyophilized rhodamine actin used contains

groups of actin monomers that have not completely dissolved, causing them to behave as

potential nuclei. Figure 2-14 shows how the actin filament density is decreased by increasing the

resuspension time of the lyophilized rhodamine actin, allowing for the breakdown of the

aggregates of actin monomers.

In an attempt to control the polymerization further, profilin was mixed in with the

polymerization assay at concentrations between 1 and 20 pM. On surfaces treated with

NEM-myosin, profilin clearly had an effect on the filament density near the surface (Figure

2-15). The elongation rate of filaments exposed to varying levels of profilin decreased only









slightly as the profilin concentration increased (Figure 2-16). This indicates that the decrease in

filament density near the surface is mostly due to the sequestering of monomers of actin, thus

preventing nuclei from forming. The decrease in elongation rate may have only a slight effect on

the density. Although the decrease in density was apparent on NEM-myosin surfaces, when

attempting to use profilin in an assay with stamped surfaces, the amount of labeled actin

monomers that did not incorporate into filaments created a high background on the surface,

making visualization of elongating filaments over time difficult to achieve.

2.4 Discussion

The data presented demonstrates that microcontact printing can be used as an effective way

for guiding the polymerization of actin filaments. By printing NEM-myosin tracks on glass

surfaces, actin filament binding was confined to certain regions of a substratum and aligned by

controlling the path of filament elongation. Smaller track widths provided the greatest degree of

alignment by increasing the frequency of elongating ends encountering the track boundaries,

whereupon the filament elongation direction tended to preferentially align with the edge of the

NEM-myosin tracks, and alignment appeared to be confined primarily to the track boundaries.

The degree of alignment, but not the polymerization rate, depended on the concentration of

NEM-myosin used to prepare the stamp, with the lowest concentration (0.1 PM) generating little,

if any, alignment. Notably, this lower value is near the minimum concentration needed to

provide a monolayer on the PDMS stamp (20), hence it is likely that, in this case, less than a

confluent layer was transferred upon printing.

2.4.1 Mechanism for Filament Alignment

Taken together, our results can be explained by the following alignment mechanism

(Figure 2-17). Nascent filaments are captured on the track surface and elongate on the

NEM-myosin. As a filament elongates, the newly polymerized portion is quickly captured by









the myosin surface until the filament end reaches the NEM-myosin track boundary. The

filament continues to grow across the boundary while unattached to the substratum and becomes

capable of undergoing larger thermal undulations. These undulations allow the filament to be

recaptured to the track in a more aligned direction, provided the angle at which the filament

encounters the track boundary is sufficiently small, and the myosin surface density is sufficiently

large to allow NEM-myosin in the track to rebind the undulating filament end.

Clemmens, et al. previously demonstrated a similar tendency of microtubules gliding

along a kinesin-coated surface to align at a chemically-defined border (105). They found that

microtubules began to undulate as they crossed a border from kinesin-rich to kinesin-poor

surface regions and that their fluctuations were sufficient to re-align the gliding microtubules at

small approach angles. Furthermore, Sundberg, et al. observed a comparable trend when

analyzing the guidance of gliding actin filaments with respect to their approach angle on myosin

tracks with a chemically-defined border (15). While similar, the mechanism in our study differs

in that the filament lengths are bound and do not slide relative the substratum, and the elongating

filament end moves by undulating about a fixed mean position determined by orientation and

position of the immobilized portion of the filament.

Our findings suggest that at narrower track widths than measured here, even greater

alignment would be achieved, with greatest alignment occurring when the myosin track widths

approach the filament width, similar to what was previously observed with sliding microtubules

on kinesin tracks (provided that the track of kinesin was no wider than the microtubule itself)

(22). Also similar to our observed effect of track edges on elongation direction, Nicolau, et al.

observed an increased alignment of (non-elongating) actin filaments gliding by ATP-dependent

myosin translocation near the boundary of regions with different myosin density (19).









2.4.2 Potential and Applications

Our results indicate that filament-binding tracks are a viable method for guiding the

direction of filament elongation on surfaces in vitro. A potential application is laying

single-filament tracks for myosin-based transport of nano- or microparticles (i.e., "molecular

shuttles") (10). One advantage of this technique over other methods for depositing filaments on

patterns (e.g. electrophoresis and flow) is that the polymerization of individual filaments could

be potentially guided to follow tracks with complex patterns (e.g., with curves and bends,

without sharp angles), thereby achieving more complex systems and networks of

myosin-mediated transport. Moreover, after the initial segment is deposited (perhaps with

position and orientation controlled by flow or electrophoresis (23, 32, 109)), the polarity of the

generated filament track would be controlled by its (+)-end-only elongation, thereby ensuring the

proper orientation for unidirectionally-guided myosin-based shuttles.

F-actin binding tracks can potentially guide elongation of individual long filaments or

multiple interconnected shorter filaments over long distances (hundreds of microns) for

long-distance transport of nano- and micro-scale cargo. The filament density depends on the

filament nucleation rate and can be controlled biochemically (e.g., by adding profilin). We have

shown that profilin reduces the density of actin filaments near the surface with little effect on the

elongation rate. Optimization of the microcontact printing procedures and actin filament

polymerization assay can provide control over not only filament density, but also filament

elongation rates, total length per filament, and non-specific interactions between filaments and

the low-affinity surfaces (BSA and glass).

A second class of potential applications for oriented filament elongation could be the

guidance of molecular shuttles bound to the elongating filament (+)-ends, rather than relying on

myosin-based transport of gliding filaments bound to cargo (4, 7). End-tracking proteins are









capable of linking the elongating actin filament end to a particle and facilitating insertional

polymerization of the tethered end (25-27). Because elongation is guided by filament

undulations and filament binding to the track, we would expect similar guidance of elongating

filaments with or without particles bound to the filament ends, provided the particles are small

enough not to interfere with the undulating end.

In summary, guided elongation of free filament (+)-ends represents an additional tool for

transporting molecular shuttles, either by laying tracks in specific patterns for myosin-based

shuttles, or by guiding propulsion of particles bound to filament (+)-ends. Further investigation

of surface properties, including other methods of binding filaments, density of filament-binding

molecules, and varying track shapes, may provide additional control over the alignment and

guidance of polymerizing actin filaments. This approach, which relies on filament elongation

rather than filament transport, should complement other published methods for actin alignment

and actin-based molecular shuttles (6, 23-24) and broaden the methods available for transport in

bio-based nano-devices.










59.01 nm


0 nm
15 [nm

C
13.5












0 7
Horizontal Displacement (pim)

Figure 2-1. Images of microcontact-printed myosin tracks on a glass coverslip. A) TIRF image
of NEM-myosin track (brighter region slightly right of center) delineated by
nonspecific adsorption of fluorescently-labeled actin monomers to adsorbed
NEM-myosin (scale bar = 10 [tm). B) Topographic AFM image of track edge
showing uniformity of NEM-myosin coverage. C) Line-scan of vertical displacement
versus horizontal displacement from plot B. The height of the step-edge was found to
be approximately 8 nm and the peak-to-valley height variations in the NEM-myosin
region were found to be on the order of 4.4 nm.





































Figure 2-2. Time-lapse image of actin filaments on a BSA-stamped surface containing no
pattern. Filaments did not bind to the surface, and many fluctuated into and out of the
200 nm TIRF region. The first frame is 20 seconds after the time-lapse began, which
was approximately 1 to 2 minutes after polymerization was initiated (0.75 giM actin,
30% rhodamine-labeled actin, scale bar = 10 tm).


45 sec












95 sec























Figure 2-3. Total internal reflection fluorescence microscopy images of elongating actin
filaments on NEM-myosin tracks of approximately 3.7 tm (A) and 19.5 tm (B)
widths. Images were taken approximately 48 min and 22 min after initiation of
polymerization, respectively (1.5 tM actin, 15% Alexa 488-labeled actin, scale bars
5 tm).


Figure 2-4. Filaments in BSA region undergo large thermal undulations compared to filaments
bound to NEM-myosin region. The NEM-myosin region, located in the top half of
the field-of-view as indicated by the brighter background, binds filaments to the
surface as they elongate, while filaments in the BSA-passivated region (bottom half)
continue to leave and enter the field-of-view, indicating that they are not bound to the
surface (scale bar = 5 tm).
























Figure 2-5. Total internal reflection fluorescence microscopy images of undulating ends of
elongating filaments. A) Images from a time-lapse sequence for a filament end
elongating within a NEM-myosin track (time interval = 195 sec; dashed-line indicates
track edge). B) Composite of images from A, indicating small undulations away
from the ultimate filament trajectory when elongating on NEM-myosin. C) Images
from a time-lapse sequence for a filament elongating across the track edge and into
the BSA region (time interval = 100 sec). D) Composite of images from C, indicating
larger undulations of the filament end over the non-binding BSA surface (scale bars =
2 im).











Figure 2-6. Filament elongating passed the track edge at small 0. A filament binds initially to
the NEM-myosin track (top half of field-of-view) and begins to undulate, binding
down as it elongates, until it eventually approaches the track edge. The filament
leaves the track edge at small 0 and undulates over the BSA-passivated region
(bottom half). The undulations are eventually large enough that the filament
encounters the NEM-myosin track and rebinds in a way that aligns the filament with
the track edge (scale bar = 2 nm).











1 -

0. -

0.6

S0.4 -

0.2

v 0 -4I I I -I
-0.2 1 0.2 0.3 0.4 0.5 0.6

-0.4 -

-0.6

Filament Density (pim filament length/pm2)


















Figure 2-7. Filament alignment as a function of filament density on tracks. Each data point on
the graph represents the average alignment of each field-of-view for the 20 [tm wide
tracks as a function of the density of all filaments within the track boundaries. Any
density higher than 0.4 tm filament length/ tm2 was eliminated due to the potential
of filament-filament interactions interfering with the alignment of the filaments on the
tracks. B) A field-of-view with a low density of filaments allows observation of
interactions between filaments and the modified surfaces only. C) A field-of-view
with a high density of filaments (density > 0.5) inhibits analysis of the sample and
may include other interactions. The tracks in the high density sample are between the
first two lines and the second two lines (scale bars = 10 [tm).



















B
0.5
0.4 -
^ 0.3
-ll
-'
S0.2

0.1 -r

I I I I | I I I I | I I I I | I I I I I
-0.1 10 15 20
Track Width (pm)

Figure 2-8. Effect of track width on filament alignment. A) Example of filament segmentation
to estimate orientation angle 0 with track edge. Left: Original TIRFM image; Middle:
Same image showing filament segments; Right: Enlargement of boxed region in
middle image showing how 0 was determined for each individual segment. Scale bar
= 2 km. B) Solid squares represent the degree of filament alignment with
NEM-myosin tracks versus track width. The error bars represent the weighted
standard error among independent samples for each track width. Significant filament
alignment was present in the three narrowest track widths (3.3 km: n = 16, p = 0.005;
4.3 am: n = 36, p < 0.0001; 5.7 km: n = 14, p = 0.042; 10.7 km: n = 17, p = 0.11;
15.1 km: n = 19, p = 0.14; 20.2 km: n = 10, p = 0.051). Open squares represent the
alignment of filaments within approximately 1.5 am of the track edge (4.3 km: n =
36, p < 0.0001; 5.7 km: n = 14, p = 0.006; 10.7 km: n = 14, p = 0.001; 15.1 km: n =
13, p = 0.036; 20.2 km: n = 7, p = 0.038). C) Time-lapse sequence showing the
alignment process for an elongating filament (+)-end at the track edge (scale bar = 2
kim).





























Figure 2-8. Continued


80 sec


160 sec











100 n-=


80
n=1l
.--o
n60 n=15



i 40
4



o n=25
20


0 5-0n=15 n= 20

0-15 15-30 30-45 45-60 60-75 75-90

Filament Angle (degrees)


Figure 2-9. Histogram showing the fraction, of filament ends that rebind to tracks after their
elongating ends cross track boundaries with varying initial angle of incidence with
track edge. Error bars represent + standard error = (f(1-f)/N)12, for N total filaments
observed within each range of angles (105). Note: No filaments crossing the track at
0 > 600 were observed to rebind to the NEM-myosin track.










'. .. -6 m
r G "" P'/ --" "- "- "| "
woo04 q ,-i


02 'I- u1 .
0.8 IN a Wlr N" -'f M IMP a a 1



IN IN I.
1N 1 IN




Sdi g.i T da ac m




to align at the track edge if their alignment angle is sufficiently small. Note that the
Distance rme EdgeaJa -a 10













highest concentration of segments occurs between 0 and 3 m of the track edge (n =
4374 segments for all track widths at 1 EM NEM-myosin concentration).
4374 segments for all track widths at I CLM NEM-myosin concentration).













0.4
.4 n= 14
=p =0.0004
^ 0.3 n=17
ccD n= 46
C- p= 0.015 p<
0 0.2 < 0.
S -n=101
0.1 0.39
0


0 0.5 1 1.5 2 2.5
-0.1
NEM-Myosin (pM)



B 5 n=12 n= 21
n= 15 E
4 i n=20
41

S3
.2 1.: 2
I



0 0.5 1 1.5 2 2.5


NEM-Myosin (pM)

Figure 2-11. Dependence of filament alignment and elongation rate on the concentration of
NEM-myosin used for microcontact printing (average track width = 4.6 + 0.6 nm).
A) Alignment appears to decrease for 0.1 gM NEM-myosin treatment condition. The
error bars represent the standard error among independent samples for each myosin
concentration. All treatments except 0.1 kM NEM-myosin provided significant
alignment of filaments (n = number of tracks, p = one-tailed p-value). Additionally,
the 2 kM and 0.1 gM samples were statistically different from each other (p = 0.021).
B) Filament elongation rate is insensitive to changes in NEM-myosin concentration
used in printing (0.75 aM actin, 15% rhodamine-labeled actin; n = number of
filaments that instantaneous velocities were measured, p-value = 0.17 from a one-way
ANOVA). The error bars represent the standard error from a least squares regression
analysis.











1 V' .mp W: *0q. : .*
0.8i W ,q No
0.6 ,
S 0.4 *.
S 0.2 -*
a *U
-0.2 1 t. a. m 1.5 i a 2.5 3 3.5
Em, M
-0.4 E -m 0. m m. s s
-0.6 M* *e *, *
-0.8 i .. *. m
-.1 *. ". v B .oi .- .' ..
Distance from Edge (rnm)

B
1 LT.m1?St.'. J $ 0. 8-** **.v >.ft 0' R
0.6 '. *
A 0.4 o *m& *'
< r ifg
o..+ 02 ., ." : *
0.2 r ,
0 0
V -0.2 *"mm.5 2.5 3 3.5

-0.4 mm m m ". r a
-0.6 00" M M
me .* a IS
-0.8 **a .* M..< .
-1 t Jh*L' ./i.l..i Joy *. *
Distance from Edge (plm)


Figure 2-12. Scatter plots showing the alignment of individual filament segments (independent
of length) at varying NEM-myosin concentrations as a function of the distance from
the track edge. A) The 0.1 gM NEM-myosin treatment had the least number of
segments, and the alignment does not seem to have a strong dependence on the edge
of the track (n = 365 segments). B) NEM-myosin concentration of 0.5 gM has a
slightly higher density of aligned filaments (n = 524). C) 1 kM NEM-myosin has a
similar trend to Figure 2-10 (n = 1583). D) A majority of segments near the edge are
highly aligned in the 2 gM NEM-myosin concentration sample (n = 487).

















A


0
l)

v
V










D


1
0.8
0.6
0.4
0.2
0
-0.2 e
-0.4 \
-0.6
-0.8
-1




1
0.8
0.6 1
0.4
0.2 -
0
-0.2
-0.4 -


-0.8
-1


*
.*


. ..




*

Eim. Et

Distance


2 -
.i.
S ,m U

j1E* U. U *
U f Uo E


e from Edge (lim)


"'> .E. '..


* D '..r =. = -


Distance from Edge (um)


3 3.5


Figure 2-12. Continued




























Figure 2-13. Filaments accumulate in the BSA region of the stamped surface, preventing
binding from occurring in the track region. Surface stamped with 2 [tM NEM-myosin
treated PDMS stamp. Image taken 11.5 min after initiation of polymerization
(scale bar = 10 atm).


Figure 2-14. Incubation time of lyophilized rhodamine actin can help to control the density of
filaments on the surface. Surfaces have been incubated with NEM-myosin for 1 min
(no stamping) followed by BSA. A) Lyophilized rhodamine actin resuspended and
incubated for 3 hours produces a large network of filaments within 2.5 min of
polymerization. B) Resuspension and incubation of lyophilized rhodamine for 3 days
before used in a polymerization assay visibly decreases the filament density on an
NEM-myosin surface (time = 6.5 min). Both samples contain 1 [tM actin with 15%
labeled (scale bars = 10 [tm).
































Figure 2-15. Increasing the concentration of profilin visibly decreases the density of actin
filaments on NEM-myosin treated surfaces. A) 0 aM, B) 1 gM, C) 5 aM, D) 10 aM,
and E) 20 aM profilin added to the polymerization assay before initiation of
polymerization. Images were taken approximately 15 minutes after initiation of
polymerization (scale bar = 5 pm).













25
n= 19



*O .3 15 -K n=21
-= n=31 n=13
0 10 U

5

0 I

0 5 10 15 20

Profilin (piM)

Figure 2-16. Elongation rate of actin filaments as a function of profilin concentration.
Elongation rate of filaments decreases slightly with increasing profilin concentration.
Error bars represent standard error based on a least squares analysis, and n refers to
the number of filaments for each concentration observed. Elongation rates were
obtained for each condition from a best-fit line of change in length versus change in
time.










































Figure 2-17. Illustration of the likely mechanism for actin filament alignment on NEM-myosin
tracks. Elongating filament ends first encounter and then undulate over the track edge.
The increase in filament length over the track causes larger undulations to occur.
Finally, the filament either is recaptured by the track for smaller 0 (A), or continues to
elongate away from the track for larger 0 (B).









CHAPTER 3
SIMULATING ACTIN FILAMENT ELONGATION ON MODIFIED SURFACES

3.1 Introduction

We have developed a simulation that allows us to predict the effect specific actin and

surface parameters (filament length, filament persistence length, myosin surface binding

probability, and patterned surface track width) have on filament alignment. These simulations

complement our experimental results and aid in designing patterns to guide actin filament

elongation in applications. Mathematical modeling of thermal undulations of actin filaments in

solution (with both ends free) has been previously reported (49). We simulated the thermal

excitations of the bending modes of the undulating unbound elongating filament end (with a

fixed (-)-end) to generate realizations of the filament shape (49, 108). These simulations allowed

us to examine the effects of total filament length, binding probability, and persistence length on

the probability of filaments rebinding after crossing a track boundary. We further investigated

the effect of NEM-myosin track width, binding probability, and persistence length on the overall

alignment of filaments.

3.2 Methodology

The bending energy, E, of a filament undulating around a straight shape is used to

determine the fluctuations of the filament by calculating the shape of the filament at an

instantaneous point (Equation 3-1) (108).

LkT L
E = (0'(s))2ds (3-1)
2

Bending energy is a function of the persistence length of an actin filament, X, the length of the

filament segment, L, the Boltzmann constant, k, the temperature, T and the tangent angle, 0, at

position s on the filament. The tangent angle can be written as a Fourier series (49), with each









mode obeying the boundary conditions: 0(0)=0 (fixed end) and 0'(L)=0 (free end) (Equation

3-2).

N
O(s)= asin((n +)~) (3-2)
n=0

where N is the number of modes, an is the amplitude of the wave function for modes n. The

integral of (0'(s))2 from s = 0 to s = L was solved for from Equation 3-2 and substituted in

Equation 3-1 resulting in Equation 3-3.

SkTt 2 C
= a (n+ ZE (3-3)
4L n= n=0

According to the equipartition theorem, the average energy for each mode is equal to kT/2,

allowing us to set the energy equal to this value (Equation 3-4).

kTTt,, 2 kT
< E >= (n+ ) < a >=k (3-4)
4L 2

Rearranging Equation 3-4, we solved for the mean square amplitudes as a function of n, L and X

(Equation 3-5).

2 2L
< a >= (3-5)
A t(n + 1)

Undulations of the free end of a filament were simulated by providing random values for

the amplitudes an from a Gaussian distribution (MATLAB function RANDN) with mean zero

and variance given by Equation 3-5. The instantaneous filament tangent angles, 0(s), were then

determined by substituting the generated amplitudes into Equation 3-2. The realized function

0(s) was converted into x- and y-coordinates by Equations 3-6 and 3-7.


Ax = cos((s)) (3-6)
As









Ay = sin((s)) (3-7)
As

The coordinates x(s) and y(s) were finally obtained by numerical integration (trapezoid rule) of

Equations 3-6 and 3-7.

Elongation and binding of the filament on a filament-binding surface was simulated as

follows. The free end of the filament, with the initial length taken as 0, increased in length by As

over each time increment, At (unless otherwise specified, As =0.01 im and At = 1 s). The

probability of attachment along the unbound undulating segment length was assumed uniform

along the filament length and constant in time, such that the probability of binding was KpLAt,

where the parameter Kp is the binding probability per unit length, per unit time (im-1xsec-).

Therefore, within the simulation, binding of a filament with length L occurred in time increment

At when MATLAB function RAND, which generates uniformly distributed random numbers on

[0,1], generated a random number < KpLAt. If the filament binds, the position of binding sl is

then determined from a uniform distribution (again using MATLAB function RAND) along the

length of the free end [0, L], and the resulting shape of the newly bound filament segment is

assumed to be fixed and is taken as the instantaneous filament shape for that region [0 si] (based

on Equations 3-6 and 3-7). After binding, the bound position on the filament resets as s = 0 and

L is reset to the new length of the unbound filament end.

Filaments were simulated on patterned surfaces with binding and non-binding regions by

allowing binding to occur only on the filament-binding regions of the surface. The filament was

assigned an initial position within the track, an initial angle between 00 and 900, and a final total

length between 1 and 16 [im, all based on a random number generator (MATLAB function

RAND, unless otherwise specified). In addition, the track width of the NEM-myosin, the









number of modes, the binding probability, Kp (lm-1xsec-1), and the persistence length, A (ilm)

was set by the user. The Appendix contains the MATLAB code with comments.

3.3 Results

3.3.1 Description of Simulated Filament Elongation

To first confirm that the simulations agree qualitatively with our experimental observations

of undulating filament ends near track boundaries, we looked at the alignment of elongating

filaments restricted to the track edge (see Figure 2-9 for the experimental results). Filaments

were initially bound at their (-)-end 0.01 [lm from the edge of the track, and the initial angle was

determined to be between 00 and 900 by a random number generator in MATLAB (function

RAND). The initial position of 0.01 [lm from the track edge was used to ensure that filaments

crossed the track edge at some point during their elongation, even at small angles. The initial

angle of the filament was assumed to be the angle of incidence, or the angle at which the

filament crossed the track boundary. Filaments were then set to elongate at a rate of

0.01 [lm/sec.

The position of the filament (+)-end with respect to time shows the fluctuations of the

filament as it was elongating (Figure 3-1). Figure 3-1A clearly shows the decrease in

fluctuations of an elongating filament end due to the decrease in the length of the free filament.

The position at which the decrease in fluctuations occurred corresponds to the position on the

final filament where binding occurred (Figure 3-1B). Figure 3-1C shows the fluctuations of a

filament that never rebinds to the track. These fluctuations continued to increase in magnitude.

Figure 3-1D shows the unbound filament's instantaneous position at the end of the simulation.

We analyzed the persistence length of filaments using Equation 3-8 (48) to confirm that the

measured value of the persistence length, A, was comparable to the input value.










C(s, = cos (es, +s )- s = exp (3-8)
2X

To do so, we pooled data from 240 simulated filaments and estimated the angle between points

at distances 0.01 .im apart on the filament. Because we had a large sample of filaments to

analyze, we only used the case where sj = si+l. We then plotted the average

In(cos(0(s, + s )- (s ))) versus sj (for sj = 0.01 to 2 pm) and found the slope of the best-fit line

with an intercept set to 0. The calculated persistence length of approximately 12 .im

(R2 = 0.996), matched reasonably well to our input value of 10 im. At a lower resolution

(0.1 [m segments) with the same conditions, the persistence length for 1750 filaments was

approximately 14 [m (R2 = 0.998). Both of these values correspond to filament shapes

calculated with 2 modes. When the number of modes was increased to 10, the persistence length

was calculated (at the lower resolution) to be approximately 13.5 rim, showing little effect of the

number of modes on the persistence length of the filament.

3.3.2 Probability of Filament Rebinding

From our experiments (cf. Chapter 2), we expected filaments with smaller angles of

incidence to have a higher probability of rebinding to the track edge (Figure 2-9).

Experimentally, many characteristics of the filaments were not constant, including the lengths of

filaments, the angles that the filaments crossed the track edge, and the position of the filaments

with respect to the position of the track edge. For our first set of simulations, we kept the initial

filament position the same for every filament and then calculated the probability of the filament

rebinding once it crossed the track edge as a function of the angle of incidence. Error bars on

graphs showing the probability of rebinding were calculated as described in Figure 2-9

( standard error = (f(1-J)/N)12)).









Figure 3-2 shows the effect of decreasing the time, At, of the simulation from 1 to

0.5 seconds with a constant elongation rate on the probability of the rebinding of 3.2 gtm long

filaments (average length of filaments for our experiments). As expected, there was little

variation in the dependence of the rebinding probability on the incidence angle. The largest

difference occurs between 200 and 300, which is most likely a region of higher variability, based

on the larger error bars. We next looked at the effect of the rebinding probability as a function of

the number of modes used to simulate the filament shape. Figure 3-3 shows very little variation

and no apparent trend between the rebinding probabilities for filaments with shapes determined

by 1, 2, 3, 5 or 10 modes. For this reason, we set the number of modes to 2 for all other

simulations.

Consistent with expectations, we found that the probability of rebinding increased with the

final filament length (Figure 3-4A). We looked at three filament lengths representing values

similar to the minimum (1.6 gim), the average (3.2 gim), and the maximum (6.4 gim) lengths from

our experiments. Filaments with angles less than 700 had a chance of rebinding if they were

allowed to elongate to 6.4 gim, and filaments with angles less than 30 rebound to the track 100%

of the time. Similarly, filaments that were allowed to elongate 3.2 gtm and 1.6 gtm had a

probability of rebinding to the tracks at angles of incidence less than 400 and 300, respectively.

Every filament that elongated past the track at an angle of 100 or less and was allowed to

elongate 3.2 gim, rebound to the track. Each filament length had the same decreasing probability

trend with increasing angle of incidence, but the final length determined at what angles the

decrease of probability began and ended. Binding probabilities were similar for the 600 at

6.4 gm, 400 at 3.2 gim, and 300 at 1.6 gtm filament lengths as well as at 400, 200, and 10,

respectively.









We also combined all of the data for all three filament lengths, which contained 300

filaments for each length, creating an average filament length of 3.7 [im for 900 total filaments

(Figure 3-4B). The trend and values for the combined data is very similar to that found in our

experiments. Experimentally, no filament over 600 was found to rebind. In our simulations, that

value was one iteration higher at 700, most likely due to the larger number of longer filaments in

the simulations.

Variations in the binding probability constant, Kp, as expected, had somewhat of an effect

on the rebinding probability (Figure 3-5). Constants of 5 and 1 im-1xsec-1 had similar results,

indicating that the saturation of the NEM-myosin may occur around a Kp of 1 im-1xsec-1. In

both cases, these Kp values allowed for rebinding to occur at angles less than 600, with all

filaments less than 300 rebinding every time. A Kp value of 0.5 im-1xsec-1 resulted in similar

rebinding probabilities at 5 and 1 rm-1xsec-1, with very slight differences (no filaments rebound

between 500 and 600, and a few filaments did not rebind between 200 and 300).

Filament binding probability constants of 0.1 and 0.05 im-1 xsec- had a similar effect as

the other three values up to 200 to 300, at which point the rebinding probability was significantly

lower than the rebinding probability for the higher Kp values. The rebinding probability of

0.05 m-1 xsec-1 also had a significantly lower rebinding probability than 0.1 im- xsec-1 in this

range. All filaments, regardless of their Kp value, had a 100% chance of rebinding if the angle

was less than 100. Finally, we looked at the effect of the persistence length of the filaments on

the rebinding probability (Figure 3-6). As expected, the rebinding probability decreased as the

persistence length increased, indicating that the rebinding probability is directly affected by the

stiffness of the filament.









3.3.3 Alignment of Filaments

In our next set of simulations, we varied the track width, allowing the filaments to be

initially placed in a random position within the track. Similar to our experimental results,

increasing the track width decreased the alignment of the filaments (Figure 3-7). We once again

varied the number of modes to make sure that two modes were sufficient. We found that the

number of modes had no effect on the alignment of the filaments as a function of track width

(Figure 3-7A). Figure 3-7B shows that filaments within 1.5 [m of the track edge have a higher

average alignment than the average alignment of all filaments. This is in agreement with our

experimental findings (Figure 2-8B).

This alignment does decrease slightly with track width due to a combination of effects.

First, the same number of filaments were tested for each track width (n = 1000) and their initial

positions were evenly distributed within the track. Therefore, the fraction of filaments with

initial positions within 1.5 [m of the edge decreases as the track width increases. In addition, as

the track width increases, filaments that elongate to 1.5 .im within the edge must have decreasing

alignments to reach the edge with lengths between 1 and 16 im. Filaments must have a

component perpendicular to the track edge, and the only way for filaments to reach the edge as

the track width increases without increasing the final length is to decrease their alignment with

the edge of the track. Once again, these results show no dependence of alignment on the number

of modes.

Next, we varied the binding probability constant, Kp, between values of 0.001 and

1 m-1 xsec-1 and found no dependence on the overall trend or values of the alignment as a

function of track width (Figure 3-8). This indicated that Kp did not have an affect on the

alignment at these values, which may correspond to our experimental values above 0.5 iM

NEM-myosin (Figure 2-11A). Furthermore, the actual persistence length of bound filaments did









not depend on Kp, with estimated values ranging from 13.3 to 14.9 [m with no apparent trend.

These values were comparable to the persistence length calculated for the same discretization

resolution with no binding (14 pm), further indicating that binding did not affect the persistence

of bound filaments..

We next looked at even lower values of Kp (0.0005 and 0.0001 im-1xsec-1) for a track

width of 4 rm to compare to our experimental analysis (Figure 3-9). We found that the

alignment began to decrease for these Kp values. The difference between the alignment for Kp

values of 0.001 and 0.0001 im-1xsec-1 is comparable to the difference between the values of

alignment for 0.1 and 0.5 iM NEM-myosin in Figure 2-8A. However, in our simulations,

filaments were more aligned than in our experiments. We also observed a decrease in alignment

at higher Kp values (0.01, 0.1 and 1 im-lxsec-1). At these Kp values, the probability of rebinding

to the tracks is high, however, once bound, the filaments may bind too often to fluctuate and

realign with the track edge, causing the alignment to decrease. Experimentally, we may not have

seen binding probabilities this high. In addition, we may have reached surface saturation, a

factor that was not taken into account in our simulations. We also determined that the

persistence length of the filament (Figure 3-10) had little effect on the alignment of filaments as

a function of track width. Slight differences between the alignments of filaments with varying

persistence lengths at lower track widths allude to a decrease in alignment with an increase in

persistence length.

3.4 Discussion

Our simulations have aided in interpreting our experimental results and in confirming the

microcontact printing as a means to guiding elongating actin filaments. The simulations also

provide a useful tool for designing patterns and experimental conditions for optimal filament

guidance. Furthermore, our simulation can be applied to other types of filaments, bundles of









filaments, and filaments with attached particles. We have replicated the experiments from

Chapter 2 and have determined further parameters and limitations of the system. Filament

lengths, NEM-myosin surface density, and persistence length, in addition to the angle of

incidence, affect the probability of a filament rebinding to the edge of the track.

Specifically, longer filaments have a higher probability of rebinding and also rebind with

higher angles of incidence. As we expected through our mechanism based on experimental data

and as our simulation confirmed, as the filament increases in length (passed the track edge), its

probability of rebinding, which is based on length, increases. For this reason, in applications, it

is important to control the length of the filaments. With fewer filaments, each with greater

length, alignment may be increased.

Also, as expected, the higher binding probability constants cause the rebinding probability

to increase, but only within a certain range of incidence angles. As found with our experiments,

there is a surface saturation point, and we found this in our simulations that recreated the

rebinding probability, with little difference between 0.5, 1 and 5 im- xsec-1. This lack of effect

on filament alignment suggests that the angle of incidence and the final filament length have a

stronger effect on the probability of rebinding than the density of molecules on the surface. If a

filament leaves the track at an angle greater than 700, the persistence length of the filament limits

the filament's ability to bend enough to sample the NEM-myosin track surface. If a filament is

too short, it also may not have enough energy to bend back to the track.

Figure 3-5 shows that the only angles of incidences that are affected by the binding

probability constant were in the range between 200 and 400 (persistence length = 10 rim, total

filament length = 3.2 pm). This small range of angles may explain why the binding probability

had little effect on the overall alignment of filaments evenly distributed over the entire track









width. These results support our mechanism proposed in Chapter 2, indicating that the filament

thermal undulations are responsible for the alignment of the filaments on binding tracks, and that

only very low surface densities will affect the rebinding and alignment of filaments. Figure 3-9

demonstrates that at Kp values on the order of 10-4 im-1xsec-1, small effects are seen on the

overall alignment of the filaments.

We were also able to vary the persistence length of filaments and study the effect on

filament alignment. Filaments with longer persistence lengths were less likely to rebind at

higher angles due to the decrease in the magnitude of their fluctuations. However, filaments

with longer persistence lengths had slightly higher overall alignments on tracks with smaller

widths (< 10 gm ). Therefore, in applications with filaments for longer persistence lengths, a

filament with a small initial angle will elongate with little changes in the alignment. However, if

this filament leaves the track edge, it has less of a chance of rebinding to the track. Filaments

with short persistence lengths may not align with the track edge, but may follow the track by

continuing to cross the track edge and rebind, even though elongation itself on the track is less

aligned.

We also determined that the surface does not affect the persistence length of bound

filaments, indicating that the surface density did not alter the filament shape. This result is

consistent with the experimental observation that the estimated measured persistence length of

bound filaments did not vary significantly with the myosin density (k = 21.3 [m for 0.1 kM

NEM-myosin, 15.7 [m for 0.5 kM, 14.6 [m for 1 kM, and 17.2 [m, for 2 [M). Therefore, we

assume that the NEM-myosin surfaces lock the filaments into shapes that are generated by the

thermal fluctuations of the filaments themselves.









Differences between experimental results and simulated results may be explained by the

lack of certain conditions in the simulation. One of these factors includes the interaction

between filaments, which we have not taken into account. Our simulations test one filament on a

track, whereas our experiments contained many filaments per track. Experimentally, we

eliminated surfaces that contained a high density of actin filaments. However, we could not

completely eliminate filament interactions. Filaments may block each other from following

paths that may be predicted for a single filament elongating on a track.

In addition, we did not account for any variations in the tracks of the filaments, including

the possibility of diffusion of NEM-myosin monomers from the printed region of the surface to

the unprinted regions. Binding of filaments by these diffused NEM-myosin molecules may

cause filaments that would have normally bent back to rebind to the tracks to remain in the

unpatterned region, therefore decreasing the rebinding probability. In our simulations, we

assumed evenly distributed initial binding positions of filaments within the entire track, as well

as evenly distributed initial filament angles. Experimentally, microcontact printing may cause

slight variations of NEM-myosin surface density or activity, causing a less even distribution of

filaments.











X-posi6iDn
Y- Y-psition


100 150


250 300


Time (sec)


I I


X-position (pm)

Figure 3-1. The x- and y-positions of elongating filament ends as a function of time. A) A
filament leaving the track edge at a small angle eventually rebinds, as indicated by the
decrease in fluctuations around 250 seconds. The initial angle of this filament was
190. B) The final instantaneous shape of the 3.2-rm long actin filament from A. The
thick blue line is the filament, the red circles represent binding of the filament to the
surface, and the light black line at y = 5 is the track edge. C) A filament leaving the
track at a large angle does not rebind. Fluctuations are larger in the x-direction
because the filament is elongating almost parallel to the y-axis. The initial angle of
this filament was 79. Fluctuations continue to increase with time. D) The final
shape of the 3.2-rm long actin filament from C. For both filaments, the initial
position was set to x = 0 and y = 4.99 (Kp = 0.1 im-1xsec-1, n = 2, A = 10 pm).










C 9
-- X-postion
7 --Y-positin or w


3


1 -

S0 50 100 150 200 251

Time (sec)


D


7
g.'




5.5
5

4.5
0 0.2 0.4 0.6 0.8

X-position (pm)


Figure 3-1. Continued
























76











MAs =0.01 Pm
* As = O.I- pm


0 I-


0- 10


__T__


10 20


20- 30


30- 40


40 fi


Filament Angle (degrees)


Figure 3-2. Effect of change in length (step-size of simulation) on the filament rebinding
probability. Results were approximately the same for both values of As, indicating
that the step-size has little effect on the rebinding probability (K = 0.1 m-L xsec-1, n
= 2, A = 10 rim, total length =3.2 pm). Note that for As = 0.01 rim, At = 1 sec, and
for As = 0.0005 rim, At = 0.5 sec, therefore keeping the elongation rate of the
filaments constant.


0.9 -

0 -

0.7 -

0.6

05 -

0. -

0.3 -

0.2 -


















0 n=- 1 10
0)
0.6



-o
0.4


0.2




S 0-10 10-0 2 20-30 30-40 40-50 50-60

Filament Angle (degrees)


Figure 3-3. Effect of the number of modes on filament rebinding probability. For each mode,
300 filaments were analyzed. The trend and values for all modes were similar,
indicating little effect of the number of modes on the rebinding probability
(Kp = 0.1 mrn1xsec1, A = 10 gm, total length =3.2 gm).










1-


. 0.8 -

o
0.6

-o
0.4


0.2 -


1-


0.8 -


0.6 -


0.4 -


0.2 -


0 10--3- -
I I -I I0 I
0-10 10-20 20-30 30-40 40-5C


I I 60I I80 80 90
I 50-60 60-70 70-30 30-90


Filament Angle (degrees)

Figure 3-4. Effect of total filament length on filament rebinding probability. A) For each length,
300 filaments were analyzed. The filament angles refer to the initial angles assigned
to each filament at the start of the simulation, which correspond to the angle at which
the filaments crossed the track edge, or the angle of incidence. The trend for all
lengths are the same, however, the rebinding probability of the longer filaments began
to decrease at a higher angle of incidence. B) The filament lengths from A were
combined for an average filament length of 3.7 gm (K = 0.1 gm-lxsec-1, n = 2,
A 10 lim)..


20 -30 30-40 40-50 50-60 60-70

Filament Angle (degrees)


0-10


M 6.4 pm
I 3.2 pm
E 1.6 pm


10 20














OO.












0-10 10 20 20 30 30 -40 40 -50 50 -60
0.6
0

0.4
0
0.2




0 10 10 20 20 30 30 40 40 50 50 60
Filament Angle (degrees)



Figure 3-5. Effect of binding probability constant, Kp (im-lxsec-1), on filament rebinding
probability. For each Kpvalue (0.05, 0.1, 0.5, 1, and 5 lm-1xsec-1), 300 filaments
were analyzed. The filament rebinding probability was unaffected by the binding
probability constant except for filaments crossing the track edge between 200 and 40.
Within this range, the higher Kp values (0.5 1 and 5 tm-lxsec-1) had similar
rebinding probabilities, while the rebinding probability decreased for both Kp values
of 0.1 and 0.05 gm-1xsec-1 (n = 2, A = 10 gim, total length =3.2 gim).










MX=5
MX=10
.S 0.8 -0X= 20
--C

9 0.6

-c
-o
0.4

0.2




0-10 10-20 20- 30 30 -40 40 -50 50 -60
Filament Angle (degrees)


Figure 3-6. Effect of persistence length on filament rebinding probability. For each A, 300
filaments were analyzed. As the persistence length increased, the filament rebinding
probability decreased at angles above 100 (Kp =0.1 gm-Lxsec', n = 2,
total length =3.2 gim).









n=2
n=5
A A n=10
S..Experimental

A f

V\ A A

1 --4--


0 5


Track Width (pm)


0.5 -

0.4 -

0.3

0.2 -


A
AT-


A
!.:


A
A A
A t


U I I I I I
0 5 10 15 20
Track Width (pm)

Figure 3-7. Effect of track width and number of modes on the alignment of filaments. A) The
simulation produced the same trend found experimentally, with no dependence on the
number of modes. B) Effect of the track edge on the alignment of filaments. Only
filaments or parts of filaments within 1.5 pm of the edge of the track were included in
the calculation of the alignment. The values are mostly within the error bars of the
experimental data, with a slight decrease in alignment as the track width increases.
The number of modes did not effect the alignment of filaments.


t -


r 3










+ 0.001
A 0.01
0.1
1
-- Experimental


0.5


0.4
A-


0.3


0.2 -


0.1 -


0


10
Track Width (pm)


- A




i| z


* 6


10
Track Width (pm)


Figure 3-8. Effect of track width and binding probability constant on the alignment of filaments.
A) The simulation produced the same trend found experimentally, with little
dependence of alignment on the binding probability constant, Kp. B) Effect of the
track edge on the alignment of filaments. Only filaments or parts of filaments within
1.5 gm of the edge of the track were included in the calculation of the alignment. The
results are similar to 3-7A, with little dependence of the alignment on Kp (n = 2, A =
10 gim).


I ^A

+AL
*i *
1

ii J i, J


0.5


0.4


0.3


0.2


0.1


-I.I











0.4 -


0.3 -
A


o 0.2
v


0.1



0 I II
0.0001 0.001 0.01 0.1 1

Binding Probability Constant (im- xs-1)


Figure 3-9. Effect of binding probability on the alignment of filaments. The alignment of
filaments begins to decrease at binding probabilities with magnitudes of
10-4 gm-1xsec-1. This implies that not enough binding occurs to rebind filaments once
they leave the edge of the track. The decrease at higher Kp values may indicate too
much binding, resulting in a decrease of filament fluctuations and the subsequent
decrease of alignment (track width = 4 gm, n = 2, A = 10 gim).










0.6 -

0.5 -


o

v 0.3 -


0.2

0.1

0 -


A=5
SA= 10
,A X=20
-U- Experimental








I4^-


Track Width (pnm)


A
m-


---4f,+


U


I


Track W'idth (pm)


Figure 3-10. Effect of track width and persistence length on the alignment of filaments. A) The
simulation produced the same trend found experimentally, with a slight dependence
of the alignment on the persistence length. The higher persistence lengths produced
lower alignment values at narrower track widths (< 10 gim). At a track width of 12
lim, all three conditions produced the same alignment. B) Effect of the track edge on
the alignment of filaments. Only filaments or parts of filaments within 1.5 gtm of the
edge of the track were included in the calculation of the alignment. Results are
similar to Figures 3-7B and 3-8 B.


SAt


0.5


0.4 -


0.3 -


0.2-


I I I I I









CHAPTER 4
ACTIN-BASED MOTILITY OF LISTERIA AND PARTICLES ON MODIFIED SURFACES

4.1 Introduction

We have replicated actin-based motility with both Listeria and ActA-coated 500-nm

diameter polystyrene beads in bovine brain cell extract. NEM-myosin treated glass coverslips

were used to provide an anchor for the actin rocket tails attached to the motile particles.

Although actin rocket tails contained many filaments (- 103), providing a system much different

than single filament elongation, these surfaces were able to confine tails and control the

propulsion of the associated particles, as observed through both real-time phase-contrast imaging

and TIRF.

We found that the paths of particles attached to confined actin-rocket tails lacked large

thermal undulations (Brownian motion) relative to those particles and tails in solution. The

effects of the NEM-myosin surfaces on the persistence and elongation rate of the actin rocket tail

and the propulsion of the attached particle have been analyzed. Treatments of glass coverslips to

create patterns or channels, including microcontact printing and photolithographic lift-off of

cyclic transparent optical polymer (CYTOP), a fully fluorinated polymer that reduces

nonspecific protein interactions (110), were used to attempt to guide the path of the particles near

the surface.

4.2 Materials and Methods

4.2.1 Listeria monocytogenes Growth and Protein Purification

Listeria monocytogenes Strain Lutl2 (pactA) over expressing ActA (87) and labeled with

4',6-diamidino-2-phenylindole (DAPI), was generously cultured and prepared by Will Zeile

(University of Florida, Gainesville, FL). Listeria strains SLCC 5764 and DPL2823 were a kind

gift of Dr. Daniel Portnoy (University of California, Berkley, CA).









A truncated version of the transmembrane protein ActA with a His6 tag on the N-terminus

was purified from Listeria monocytogenes (strain DPL2723) (111). Agar plates (containing

brain heart infusion (BHI) media with agar and chloramphenicol) were streaked with bacteria,

incubated for 24 hr at 37 C, and stored at 4 OC. A colony of bacteria from the plates was added

to 50 mL of BHI media diluted in ddH20 with 10 tg/mL of chloramphenicol and incubated

overnight at 37 C while shaking at 225 rpm. The bacteria solution was then transferred to 1 L

BHI with 10 tg/mL of chloramphenicol and incubated at 37 C while shaking (180 rpm) for 6 to

8 hr (final OD600 = 0.6 to 0.8).

The bacteria solution was cooled on ice for 20 min and clarified by centrifugation at

2700 x g for 20 min at 4 OC in a JA-10 rotor (Beckman Coulter, Inc., Fullerton, CA).

Ammonium sulfate was slowly added and dissolved (Ihr) in the bacteria solution for a final

concentration of 50% (313.5 g/L) at 4 OC and was left in 4 C for 3 hr while stirring to

equilibrate. The precipitate was centrifuged at 4 OC for 30 min at 7000 x g in a JA-10 rotor and

resuspended in less than 10 mL of Hepes-KCl buffer (HKB; 20 mM Hepes pH 7.4, 50 mM KC1,)

supplemented with protease inhibitors (1 complete mini, EDTA-free tablet per 10 mL, 1 mM

PMSF) per liter of supernatant. The protein was eluted from solution using a 5 mL bed volume

of Talon Ni-NTA resin equilibrated in HKB supplemented with 250 mM Imidazole. The

fractions were dialyzed against HKB overnight to remove Imidazole from the buffer (111).

Actin was purified and labeled with Oregon green 488 carboxylic acid, succinimidyl ester

(Invitrogen, Molecular Probes, Carlsbad, CA), with the same procedure described in Chapter 2.

NEM-myosin was also prepared as described in Chapter 2.

Frozen whole bovine brains were grinded into a fine powder in liquid nitrogen and mixed

with an equal volume of sonication buffer (10 mM Tris-HC1, pH 7.5, 2 mM MgC12)









supplemented with 10 [g/mL each of pepstatin A, leupeptin, chymostatin, and 1 mM PMSF.

The mixture was dounce homogenized 30x on ice, sonicated on ice with a tip sonicator for

30 sec bursts at 25% power, and then centrifuged at 17,000 x g for 20 min at 4 OC in a Ti60 rotor

(Beckman Coulter, Inc., Fullerton, CA). The supernatant was then centrifuged at 118,000 x g for

1 hr at 4 C in a Ti60 rotor. The supernatant was recovered and supplemented with 1 mM DTT

and 1 mM ATP. The extract was aliquoted, flash frozen with liquid nitrogen, and stored at

-70 C (112).

4.2.2 Bead Preparation

500-nm diameter polystyrene beads (Polysciences, Inc., Warrington, PA) were diluted in

MOPS buffer (100 mM MOPS pH 7.0 with KOH) for a final of 0.5% solids solution. The beads

were rinsed twice by centrifuging at 16,000 x g for 5 min at 4 OC (Eppendorf 5415C

microcentrifuge), and resuspending in MOPS buffer. Bis[sulfosuccinimidyl] suberate (B S3)

(Pierce, Rockford, IL) was diluted to 20 mM and added dropwise to the beads, and the solution

was incubated for 15 min at room temperature while shaking. The BS3 solution was removed by

pelleting the beads through centrifugation (5 min, 16,000 x g) and ActA was added directly to

the beads at 200 to 250 [g/mL. The solution incubated at room temperature for 1 hr while

shaking gently. The ActA solution was removed by centrifugation (5 min, 16,000 x g), and the

beads were resuspended and rinsed twice in a 200 mM solution of glycine methyl ester in MOPS

buffer to block all remaining BS3 binding sites. Beads were rinsed once in MOPS buffer,

resuspended in brain extract buffer (20 mM Hepes, pH 7.5, 1 mM MgC12, 1 mM EGTA, 100 mM

KC1, 0.2 mM CaC12, 150 mM sucrose), and stored at 4 OC until activity of the beads decreased

significantly, which typically occurred within one week (William Zeile, unpublished).









4.2.3 Motility Assay

Motility assays consisting of extract from bovine brain cells were used to mimic in vivo

conditions for actin-based motility of Listeria and 500-nm ActA-coated polystyrene bead. An

aliquot of bovine brain extract was thawed and clarified by centrifugation at 16,000 x g for 10

min at 4 C. The supernatant was aspirated and supplemented for a final composition (by

volume) of 10% creatine kinase (CK)-ATP regenerating solution (7.5 mM creatine phosphate, 2

mM ATP, 2 mM EGTA, 2 mM MgC12, 50 [tg/mL creatine kinase), 10% protease inhibitors (10

[tg/mL each of pepstatin-A, chymostatin, and leupeptin), 10% (10 mM) dithiothreitol (DTT) and

75% clarified extract (113).

After incubating on ice for 5 min, Oregon green 488-labeled actin was added to a final

concentration of 5.7 [iM and either Listeria (final concentration = 1 to 1000 dilution of stock) or

ActA-modified beads (final concentration = 1 in 100 to 1 to 400 dilution of stock) were added in

the final extract. Actin tails formed on these beads within 30 min and propelled the particles in

solution. Motility assays used on modified surfaces were incubated in a microcentrifuge tube

until approximately 15 to 30 min after the solution was first mixed. This typically allowed

enough time for short rocket tails to form on the beads, which could then bind to the surface.

Flow-cells with cleaned glass coverslips were treated with either 2 [iM NEM-myosin

diluted in myosin dialysis buffer or 1% BSA in TM buffer (10 mM Tris, pH 7.5, 2 mM MgC12)

for 1 min, followed by 1% BSA in TM buffer to wash any unbound NEM-myosin molecules and

to passivate any exposed glass surface. A motility assay containing Listeria or ActA-modified

beads was added immediately after the BSA rinse.

4.2.4 Fabrication of Channel Devices

Using a photolithographic lift-off technique, CYTOP was patterned on glass coverslips. A

glass coverslip was photopatterned with two-coatings of NFR016, a negative photoresist. Using









an e-beam evaporator, a 50 A-thick chrome layer was deposited on the top surface of the

photoresist-patterned coverslip. CYTOP was then spin-coated onto the surface at 4000 rpm for

20 sec and was cured at 100 OC for 10 min. The slide was soaked in acetone for 5 min and

sonicated briefly at low power. This caused the regions with the photoresist layer to lift off the

surface, exposing the glass. The CYTOP layer was found to be approximately 2 tm thick based

on measurements from an Alpha-Step IQ profilomoter. PDMS stamps and CYTOP patterned

surfaces were prepared by Dr. Suzanne Ciftan-Hens (International Technology Center, Raleigh,

NC).

4.2.5 Microscopy and Analysis

Samples were observed using a Nikon Diaphot inverted photomicroscope equipped with

phase-contrast optics for up to 2.5 hr after motility assays were mixed. Images were acquired

using a digital CCD camera and analyzed using MetaMorph software. Time-lapse sequences

were 5 to 10 min long with images acquired at 15 sec intervals. Objective-based TIRF (as

described in Chapter 2) was used to image details on actin rocket tails bound to the surfaces

approximately 2 to 4 hr after motility assays were mixed.

Random fields-of-view were taken for three experiments and combined to determine the

fraction of bound actin rocket tails on BSA and NEM-myosin-treated surfaces. We only

included those tails that were almost completely in the field-of-view for approximately the entire

time-lapse sequence of 5 min. Particles and tails that were not attached to the surface were

distinguished from those that were attached because of their larger fluctuations (which also

hindered our measurements of tail length and bead velocity). A set of tails were considered to be

partially attached; they were either attached at part of the tail that was not near the bead (causing

a noisy bead trajectory) or the bead was attached to the surface but was not motile. Particle paths

were manually tracked over time using MetaMorph software.









The elongation of the actin rocket tails was calculated by measuring the change in length

of the actin rocket tail near the particle at a minimum of three time points for each tail. The

average elongation rate was calculated by pooling all of the data within each surface treatment

and determining the slope of a best-fit line (with intercept set to 0) for the change in length

versus the change in time. Measurements of tails that were not bound to the surface may have

been skewed due to parts of the tail leaving and re-entering the field-of-view. This limited our

data of tails on passivated surfaces for the final analysis. Finally, tails were traced in the last

frame of every time-lapse for both BSA and NEM-myosin treated surfaces to determine if the

NEM myosin surface had any effect on the persistence of the tail. The distance between the

end-points of the tails was squared and compared to the total length of the tail.

4.3 Results

4.3.1 Confining Particle Propulsion to the Surface

Actin-based motility was induced in vitro in cell extracts using the motility assay described

above. Both Listeria and ActA-coated 500-nm diameter polystyrene beads produced actin rocket

tails, as shown in images taken using phase-contrast microscopy (Figure 4-1). The actin tails

looked identical in both cases, despite the slight difference in size and shape between the Listeria

and the bead. The rocket tails elongated in solution, and in addition, rotated in three dimensions

over time (Figure 4-2). The rocket tails took on a variety of different conformations, many

tending to have a curved or helical shape.

In order to manipulate the path of the particles, we eliminated the rotation and thermal

motion of the rocket tails in solution by confining them to NEM-myosin glass coverslips. Actin

tails that were attached to the surface were distinguished from tails that were not attached by

their decreased Brownian motion observed in real-time phase-contrast microscopy. Figure 4-3

shows an actin rocket tail attached to Listeria on an NEM-myosin surface over time. The rocket









tail remained stationary while the bacterium was propelled forward. On a BSA-coated surface,

the actin rocket tail elongated but also changed position over time. Figure 4-4 shows similar

results for a 500-nm diameter bead attached to a tail on an NEM-myosin and BSA surface. In

4-3B and 4-4B, the tail and the Listeria or bead remained in the focal plane for the entire

time-lapse, moving only in the x- and y-directions. Many free tails also moved in the

z-direction, coming into or out of the focal plane during the time-lapse. In addition, some free

tails had part of their structure in the focal plane and the remainder above the focal plane for the

entire sample time.

4.3.2 Effectiveness of NEM-Myosin Surfaces

Many samples contained a high percentage of actin rocket tails bound to the surface, as

indicated by their lack of movement over time (Figure 4-5). The number of tails bound per

field-of-view varied, depending on the sample, the time of the assay, and the position in the

flow-cell. On NEM-myosin-coated surfaces, equal fractions of actin rocket tails attached to

500-nm diameter beads near the surface were completely bound or partially bound, comprising

-60% of the total number of tails (Figure 4-6). Partial binding occurred when a tail was attached

at certain points away from the particle or when a particle attached to the surface was not

moving. On the BSA surfaces, only one tail was attached to the surface, indicating that the

binding of the actin rocket tails was caused by specific interactions with NEM-myosin.

The movement of the free tails in solution caused the trajectories of the attached beads to

appear chaotic compared to the smooth trajectories of the particles attached to bound tails

(Figure 4-7). Particle motility in the bound case was likely controlled by actin filament

elongation within the actin rocket tail. For particles not bound (on either NEM-myosin or

BSA-treated surfaces), the trajectory was chaotic because, in addition to elongation, the rocket

tail itself was moving with respect to time. These results suggest that NEM-myosin can









effectively bind actin filaments within a large actin tail, confining the tail to the surface for some

time while continuing to propel particles attached to the actin rocket tail in a more controlled

manner. In some cases, the interaction between the NEM-myosin and the actin rocket tail was

enough to confine actin rocket tails with helical shapes, as shown in Figure 4-8. It is unknown in

this case if the actin rocket tail landed on the surface as a helix or elongated into that shape while

bound to the surface. However, the particle did continue to follow the curvature of the helical

path as its tail elongated, which is most likely an effect of the long persistence length of the

rocket tail.

Even though the entire tail remains in the same position over time for many samples, it is

likely that only some regions of the tail were attached. TIRF images show that regions of the

rocket tails were clearly bound to the coverslips (Figure 4-9), while other regions were not

interacting with the surface. Slightly above TIRF range (> 200 nm from the surface), the entire

rocket tail was observed, proving that, in many cases, the actin rocket tail was bound randomly

along its length. This implies that the entire length of the tail did not need to be interacting with

the surface for the tail to remain bound. An enlarged TIRF image of the actin rocket tail

revealed potential actin-surface interactions at the sides of the tail, where single filaments or

small bundles seemed to protrude away from the main structure (Figure 4-10). It is unclear if

these single filament protrusions were located at the bottom of the tail as well. The binding

interactions may have occurred mainly with these filaments.

4.3.3 Particle Velocity and Tail Characterization

We next looked at the effect NEM-myosin substrata had on both elongation rate and

persistence of actin rocket tails. Figure 4-11 shows a histogram of tail elongation rates on both

NEM-myosin and BSA surfaces. NEM-myosin surfaces had a narrower distribution of

instantaneous rates, whereas the BSA surfaces were more wide-spread, containing some rates









below 0 rm/sec. The change in length versus time was plotted for bound actin rocket tails on

NEM-myosin surfaces and free tails on BSA surfaces (Figure 4-12). The slope was calculated

from a best-fit line to determine an overall tail elongation rate of 0.0157 0.0005 utm/second for

tails attached to NEM-myosin surfaces and 0.0135 + 0.001 utm/second for free tails near a BSA

surface, showing little effect of the surface on the rate of particle propulsion. Accurate

measurements were difficult to obtain for the free tails due to the motion of the tails over time in

the x- and y-direction and also in and out of the focal plane. This difficulty in measurement

could imply that the free filament elongation rate reported may be lower than the actual

elongation rate due to loss of data. This may also account for the wider distribution of

elongation rates and the negative elongation rates acquired for certain samples.

The persistence of the actin rocket tails was measured by comparing the total length of the

tail to the distance between the start and the end of the tail. The ratio of distance to length of the

tails bound to the NEM-myosin surface was similar to that of free tails up until lengths of

approximately 35 rm, at which point bound or partially bound tails have a slightly higher ratio

than free tails on NEM-myosin or BSA surfaces (Figure 4-13). This implies that the

NEM-myosin had a slight effect on the final shape of the actin rocket tail and subsequently, a

slight effect on the final path of the 500-nm particles. Furthermore, the average length of the

actin rocket tails measured on the NEM-myosin surface (28.4 + 1.5 um) was statistically greater

than the average length for those on the BSA surfaces (21.8 1.7 um), with a two-tailed

p-value = 0.0196. This result indicates that tails bound to NEM-myosin-coated substrata contain

more tail length within a two-dimensional plane than free tails. In this case, the NEM-myosin

surface had a clear effect on the tail shape, eliminating the freedom of the tails to bend into the

z-direction.









4.3.4 Guiding Particle Propulsion

Microcontact printing was used to attempt to further guide the particles, similar to the

guidance of single filaments. There are many differences between actin rocket tails and single

filaments, including size and solution conditions, indicating that this method may not be as

effective for the large bundles of actin filaments. Initial attempts show selective binding of the

particles to the NEM-myosin, but confinement does not compare to the tails bound to

unpatterned NEM-myosin surface (Figure 4-14). Although tails remained near the surface, many

rotated or moved around one point of attachment, implying that short tails shown in the time

series were only partially bound to the surface. Furthermore, these tails did not elongate over

time, preventing motility of the attached beads.

Factors that may be inhibiting the actin tail-NEM-myosin interaction on the

microcontact-printed surfaces include the density of the NEM-myosin layer, conformation of

NEM-myosin molecules and the binding activity of the NEM-myosin with actin filaments. In

addition, the brain extract used contains large amounts of protein, including myosins,

tropomyosins, and actin. All of these proteins may have bound to the regions of NEM-myosin

non-specifically, blocking the binding of the actin rocket tails to the surface.

Three-dimensional surface structures using patterned CYTOP on glass coverslips have

been tested for their ability to guide 500-nm diameter beads undergoing actin-based motility.

While many actin tails were confined to the NEM-myosin-treated glass portion of the surfaces of

these devices (Figure 4-15), the ability of the device to guide the particles has not yet been

determined. Figure 4-16 shows a particle being propelled by an actin rocket tail encountering a

wall at a perpendicular angle. The particle was propelled over the wall, indicating that the height

and design of the wall were not optimal to guide the path of the particle.









4.4 Discussion

4.4.1 Mechanics of Actin Rocket Tails on Surfaces

The results obtained in this chapter suggest that surface manipulation may be used to

confine the propulsion of both Listeria and 500-nm diameter particles undergoing actin-based

motility. NEM-myosin adsorbed to glass surfaces has the ability to confine the actin rocket tails

to the substratum while continuing to propel the objects. NEM-myosin molecules bind to single

actin filaments, thus the tails must be confined to the surface by many interactions between

single actin filaments within the tail and single NEM-myosin molecules.

Based on our TIRF images, NEM-myosin may create a place for actin filaments protruding

and elongating from the sides of actin rocket tails to bind. In solution, filaments are likely

protruding from all sides of the tail. Therefore, the side of the tail facing the surface may be

interacting with the surface through these small filaments rather than filaments that are

cross-linked within the main tail. These filaments may have been side branches created by the

presence of Arp2/3 in the extract (114). If these filaments are present in vivo, they may function

by incorporating in the cytoskeletal actin network, providing an anchor for the propulsion of

Listeria in the cytoplasm. Our TIRF images also indicate that the entire length of the tail does

not appear to be bound to the surface. Tails may be confined to the surface at random places

along their length, and this may be sufficient for overall confinement of the tail. Another

possibility is the areas that do not appear directly on the surface may be cross-linked to the

nonspecific actin filaments that are attached to the surface, creating a network from the

NEM-myosin surface to the tail.

Only filaments in the tails that are contacting the substratum are available for binding to

the substratum, a fact which limits the effect the substratum properties has on the entire actin tail

and the propulsion of the particle. The most significant effect of the surface-tail interaction is the









ability to reduce the noise in particle trajectories. By controlling the trajectories, particles are

more likely to follow a designated path on a surface and move against flow or diffusion

gradients. The tail elongation rate and two-dimensional shape was not affected significantly by

the NEM-myosin surfaces. (The surface did affect the shape of the tail with respect to the

z-direction.) The rocket tails contain many filaments, all of which contribute to the overall

elongation rate and shape of the tail. Since only filaments near the surface are interacting with

NEM-myosin, it is expected the surface interactions will have little effect on these parameters.

Furthermore, the filaments protruding from the sides of the rocket tail, which are likely to be

interacting with the substratum, should not effect the elongation rate at all since they are not

elongating near the surface of the motile object.

4.4.2 Biochemical Considerations

Biochemical modifications may control certain parameters of the system, including

percentage of beads that form tails, length of tails and velocity of the particles. We have tested a

variety of extracts, including rat brain, bovine brain, and human platelet extracts. All three of

these extracts supported motility of Listeria and beads. A thorough analysis of the extracts may

be beneficial for optimization of the assay. The extracts may contain variable amounts of VASP,

Arp2/3, ATP and ADP, protease inhibitors, ADF, cofilin, profilin and actin, as well as many

unknown proteins that could aid or inhibit certain aspects of actin-based motility (115).

Cameron, et al. showed that extract dilution, in addition to bead diameter and ActA coverage,

influenced the speed of particles and the tail curvature (116). We expect these parameters will

have the same effect on beads attached to the surfaces.

Another important factor is the time-sensitivity of the 500-nm ActA-treated particles, i.e.

the same particles may behave differently as soon as one day after initially making and testing

the sample. The particles tend to aggregate and the surface concentration of ActA decreases over









time through desorption. Even though ActA was cross-linked to the surface of the particles, it is

possible that some ActA only physic-adsorbed to the beads and is aiding in tail formation and

elongation. The subsequent desorption of this ActA may be enough to decrease tail formation

significantly.

Surface binding is also dependent on the length of the tails when the motility assay is

added to the modified glass coverslips. Before actin tails begin to form, an actin cloud, or a

cross-linked actin network, surrounds the ActA-coated motile object. The bead inside this

network may make small fluctuations, eventually causing the network to break spontaneously to

one side of the bead, i.e. symmetry break (117). If the assay is added to the surface during the

actin cloud state, the cross-linked actin network may bind to the surface, trapping the bead inside

the network, ultimately preventing symmetry breaking. All of these factors combined make the

biochemistry of this system complex. It may be beneficial in the future to move from cell

extracts to single component systems (115), which could enhance the effect of the surfaces on

the actin rocket tails and particle propulsion as well as allow more control over the velocity and

curvature of the particles.

4.4.3 Considerations for Bionanotechnology

As mentioned before, each NEM-myosin molecule binds to only one filament in the tail,

leaving most of the tail unaffected by this interaction. This characteristic of actin tails could

indicate that modified substrata alone may have a limited effect on controlling the trajectories of

the 500 nm beads on the surface. With larger actin structures propelling particles, a combination

of channels and modified substrata may provide the most control over the trajectory of the

particles (105). Microcontact printing may be further investigated as a way to confine filaments

to the surface, but it is unlikely that this technique will provide the same advantages that it does

for single actin filaments. The actin rocket tail is much stiffer than a single actin filament,









suggesting that the smaller thermal fluctuations of the tail would make it difficult to bend back

and rebind to a track. In addition, the path and velocity of the propelled particle is dependent on

many filaments that may not be interacting with the surface. For these reasons, physical barriers

may be needed to provide control over the particle's path.

Further optimization of the height of the channels may prevent the particles from traveling

over the top of the channel walls. Other designs, such as undercuts may also prevent this, as

shown to be effective by Hess, et al. for the guidance of sliding microtubules (17). If the channel

walls are high enough, beads may be able to take paths that include sharp or rounded turns. In

addition, directionality of the particles may be easily obtained through the use of paramagnetic

particles or electric fields. By allowing tails to bind first and then applying a force, the particles

may change their path toward a specified direction.

Particle motility for potential use in nanodevices has been confined to methods using

single filament microtubule/kinesin or actin/myosin sliding assays, where particles are either

attached to the filament or the motor protein (2, 3, 6, 7). Bohm, et al. successfully showed

motility of large, micron-sized particles of various materials by attaching the objects to kinesin

and allowing them to move along an array of aligned, isopolar microtubules (3). We have added

another method of particle motility that exploits actin polymerization and the ActA/VASP

complex and can be useful in the future for nanodevices.




















Figure 4-1. Listeria and 500-nm diameter bead propelled by actin rocket tails. A) Listeria
placed in cell extract formed actin rocket tails at its rear. B) Polystyrene 500-nm
diameter particle treated with the surface protein ActA emulated the behavior of
Listeria under the same conditions, forming actin rocket tails. Images were taken in
phase-contrast (scale bars = 5 tm).









Figure 4-2. Rotation of a helical actin rocket tail in solution. A polystyrene 500-nm diameter
particle contained an actin rocket tail which rotated in three dimensions over time.
The arrow indicates where the tail crosses itself in the various views. The images
were taken 1 minute apart in phase-contrast (scale bar = 5 tm).










A4


" mr"
V-


9



ii) .


*% 1,j


- .


,ME,


Figure 4-3. Listeria rocket tails on NEM-myosin and BSA-treated surfaces. A) Listeria actin
rocket tail is bound to an NEM-myosin treated surface. The tail did not move over
time, as indicated by the top line. The actin rocket tail propelled the Listeria as seen
by the increasing distance between the bacterium and the bottom line over time.
Images shown were taken 75 seconds apart. B) Listeria actin rocket tail remained
free in solution when exposed to a surface passivated with BSA, as indicated by the
movement of the entire tail over the time lapse. Note there are two Listeria in this
time-lapse sharing a membrane. Images shown were taken every 45 seconds (scale
bars = 5 tm).


Figure 4-4. 500-nm diameter beads attached to rocket tails bound on NEM-myosin and
BSA-treated surfaces. A) Actin rocket tail is bound to an NEM-myosin treated
surface. The tail did not move over time, as indicated by the top line. The actin
rocket tail propelled the particle as seen by the increased distance between the particle
and the bottom line. Images shown were taken 75 seconds apart. B) Actin rocket tail
remained free near a surface passivated with BSA, as indicated by the movement of
the entire tail over the time lapse. Images shown were taken every 75 seconds (scale
bars = 10 pm).



































Figure 4-5. Fields-of-view with large percentage of tails bound to surface. A) Actin rocket tails
on Listeria bound to an NEM-myosin surface. Many tails remained in position while
elongating over the 2.5 minutes between images. B) Actin rocket tails on 500-nm
diameter beads are also bound in a high percentage to NEM-myosin treated surface.
Tails remained in position between the first image and the second image, taken 3.5
minutes apart (scale bars = 20 tm).











= 243


SBSA
* NEM-Myosin


0.9 -

0.8

0.7

0.6-

0.5

0.4

0.3 -

0.2

0.1

0


Bound


n= 49
T


Partially Bound
Partially Bound


Free


Figure 4-6. Fraction of actin rocket tails bound to NEM-myosin and BSA-treated surfaces. Only
one tail was bound to the BSA surface. Of the tails on the NEM-myosin surface, 28%
were bound and successfully propelling a particle, 31% were partially bound, and
41% were free in solution. Tails were analyzed from 20 randomly selected
fields-of-view for each surface from three different experiments. Error bars represent
+ standard error = (f(l-J)/N)1/2, for N total filaments.


n= 43
T


n 1











i

el
e


Time (seconds)


Time (seconds)


Figure 4-7. Change in x- and y-position over time for beads on NEM-myosin and BSA surfaces.
A) A 500-nm diameter bead attached to an actin rocket tail bound to an NEM-myosin
coated surface is propelled forward in a smooth trajectory. B) A 500-nm diameter
bead attached to an actin rocket tail that is in solution near a BSA-coated surface
underwent Brownian motion as indicated by the noise in the position over time.


Figure 4-8. Helical actin rocket tail confined to NEM-myosin-treated surface. Images were
taken 1 minute 15 seconds apart (scale bar = 5 im).


104 -
102 -
100 -
M
98
96 -


120
M
118 -
116
114
112
110


i


J\J1__\






































Figure 4-9. Actin rocket tails in total internal reflection fluorescence microscopy. A) Actin
rocket tail attached to Listeria was bound to the NEM-myosin surface, as shown in
TIRF. The actin rocket tail appears to have smaller bundles or single filaments
extending off the side of the main structure. B) Image of A taken in fluorescence just
above the 200-nm TIRF region, showing the entire rocket tail. Only a small portion
of the length of the tail was bound to the surface. C) Actin rocket tail attached to a
500-nm diameter bead was bound to a NEM-myosin surface. The arrow points to
where the bead was most likely located. D) Fluorescent image slightly above TIRF
corresponding to C (scale bars = 10 tm).






















Figure 4-10. Magnified image of actin rocket tail with protruding filaments. The actin tail had
single filaments or small bundles of actin extending from the main structure. Short
filaments that may have formed spontaneously in solution were also bound to the
surface (scale bar = 5 im).











60

50

S40

30 -
20
10





Tail Elongation Rate (pm x sec-1)
B
60

50

4 40
g 30
20
10

10 rn



Tail Elongation Rate (pm x sec-1)

Figure 4-11. Actin tail elongation on NEM-myosin and BSA surfaces. A) Histogram of
instantaneous tail elongation rates (frequency) observed for actin tails bound to
NEM-myosin surfaces. The peak occurs between 0.015 and 0.016 gm/sec (n = 280,
80 filaments). B) Histogram of instantaneous tail elongation rates observed for free
actin tails near a passivated BSA-treated glass surface. The peak occurs between
0.006 and 0.008 gm/sec. The wider spread of rates, including negative elongation
rates, was caused by difficulties in measuring the length of tails that were not attached
to the surface and were potentially moving in and out of the focal plane (n = 169,
58 filaments).











20 y= 0.0157x




S10 2


0 200 400 600 400
Time (sec)
B
4-1 yto determine an average elongation rate of 0.01350.0135/sec for free
-" 1 R = 0.414
0 0





0 100 200 300 400
Time (sec)


Figure 4-12. Average tail elongation rates determined from the slope of a best-fit line. A) The
changes in length of the tails attached to NEM-myosin surfaces were plotted against
time to determine an average rate of 0.0157+0.0005 ilm/sec. The two outliers in the
histogram (Figure 4-11A, > 0.05 ilm/sec) were excluded from the plot (n = 280, 80
filaments). B) Values less than 0 and greater than 0.05 were eliminated (Figure
4-11B) to determine an average elongation rate of 0.013 5+0.001 .m/sec for free
filaments near BSA-treated surfaces (n = 146, 50 filaments). An unpaired t-test gives
a p-value of 0.04, indicating that the two elongation rates are significantly different.











3500 Bound Tails

SPartially Bound Tails
3000
SFree Tails NEM-myosin

2500 Free Tails BSA


2000


1500 /
/ a.

1000 /.
/i **

500 .
-", C



0 10 20 30 40 50 60 70

Total Length of Tail (pm)

Figure 4-13. NEM-myosin surface effect on persistence of tail. Tail lengths were measured as
they appeared in the two-dimensional focal plane. The total length of the filaments
was typically longer than the distance between the end-points of the tail (r). Bound
and partially bound tails (n = 84 and n = 40, respectively) between 35 and 60 [tm long
seem to have slightly more persistence than free tails on NEM-myosin surfaces or
BSA surfaces (n = 56 and n = 197, respectively). We were able to measure more
length per filament for the bound tails due to their confinement in the focal plane.









































Figure 4-14. Stamped surfaces with 500-nm diameter beads attached to actin tails. Very faint
lines in phase-contrast were present and are enhanced by the white dashed lines.
Beads seemed to attach to the regions within the track boundaries; however,
elongation was not observed, and tails seemed to rotate or move slightly over time.
Images shown were taken 75 seconds apart (scale bar = 10 [tm).

































Figure 4-15. Actin tails bound to NEM-myosin-treated exposed glass of fabricated device.
Black regions in the images are CYTOP, while all other area is exposed glass. The
arrow points to a 500-nm diameter bead that was propelled by an actin rocket tail
bound to the glass. No tails were observed elongating inside the channels. Images
were taken 75 seconds apart (scale bar = 20 am).
























9V


,: t .. 1 : .. ..:! E ..." "






Figure 4-16. Actin rocket tail encounters CYTOP wall. A) Image of entire region of the device.
The black region is CYTOP while all other regions are NEM-myosin treated glass
(scale bar = 20 tm). B) An actin rocket tail confined to the glass surface encountered
the wall at a perpendicular angle and propelled the particle over the channel wall,
indicating that channel walls may need to be higher to guide the particles. Images
shown are at 2.5 minute intervals (scale bar = 5 tm).









CHAPTER 5
SINGLE FILAMENT ACTIN-BASED MOTILITY OF PARTICLES

5.1 Introduction

The use of rocket tails for propulsion of larger particles and bacteria is challenging in that

guiding stiff bundles of actin filaments on modified surfaces is difficult without

three-dimensional barriers (channel walls). Smaller particles (50 nm in diameter) have potential

to be propelled by a single filament or a few actin filaments while behaving similarly to

elongating single filaments without particles. As stated earlier, this may lead to guidance of

these particles using microcontact-printed tracks (Chapter 2). The challenge thus far has been

observing elongating single filaments bound to particles while persistently attached via the

ActA/VASP complex.

The only previously published observation of a single filament on a 50-nm diameter

ActA-coated bead was obtained using transmission electron microscopy (TEM) (89). Recently,

our group has observed many single filaments on 50-nm ActA-coated beads in TEM (Sturm, C.,

J. Phillips, W. Zeile, K. Interliggi, R. Dickinson, and D. Purich, unpublished), lending promise to

the possibility of single-filament bead propulsion for nanodevices. Processively elongating

single filaments have been more frequently and easily observed using formins attached to 1-[im

diameter beads (27). A bead with a diameter of 1 [m would be too large to guide on

microcontact-printed surfaces alone, but the attachment of single filaments through formins

could be applied to smaller particles.

With our recent experiments, we have shown that single filaments are attached at their

(+)-ends to small ActA-coated beads, supporting the end-tracking motor mechanism for

ActA-induced filament elongation (Sturm, C., J. Phillips, W. Zeile, K. Interliggi, R. Dickinson,

and D. Purich, unpublished). This result also verifies the potential for guiding small particles









with single filament elongation through filament end-tracking motors. To address the possibility

of single-filament actin-based motility in nanodevices, we first looked at attaching particles,

rather than tails, to surfaces. This method decreases the Brownian motion of the beads to give a

reference point for actin filament-particle interaction and allows dynamic observation in TIRF.

Once filaments could be observed consistently, the system was optimized to visualize only a

single filament or a few filaments per bead. Actin filaments were characterized by their

fluorescent intensities to elucidate the mechanism of attached filament elongation near the bead

surface. Finally, attempts were made to attach actin filaments under these conditions to

NEM-myosin surfaces to observe a motile bead. The complexity of this system was assessed

through these experiments.

5.2 Materials and Methods

5.2.1 Protein Preparations

ActA, bovine brain extract, actin, and NEM-myosin were all prepared as described in

Chapter 2 and Chapter 4.

5.2.2 Bead Functionalization

A 500-iL aliquot of 50-nm diameter silica beads (Polysciences, Inc., Warrington, PA) was

pulse sonicated 20x at low power (Benson Sonifier 450) to break apart aggregates of beads and

suspend them evenly in solution. The beads were incubated for 1 hr at room temperature with

0.2% gluteraldehyde with gentle shaking. The solution was centrifuged at 16,000 x g for 5 min

at room temperature followed by aspiration of the supernatant and resuspension of the beads in

500 [tL of 1600 tg/mL of ActA in brain extract buffer. The solution incubated for 40 min at

room temperature with gentle shaking, followed by centrifugation (16,000 x g, 5 min, 4 C).

The beads were resuspended in a 500 [tL solution of 3100 tg/mL of Oregon green 488-labeled

BSA and incubated for 1 hr at room temperature. The solution was centrifuged as before,









resuspended in 500 [L of 100 mM glycine methyl ester in brain extract buffer to block any

reactive gluteraldehyde molecules, and pulse sonicated. The solution was centrifuged and

resuspended in brain extract buffer twice to wash away any excess protein or chemicals and then

stored in brain extract buffer at 4 OC.

5.2.3 Motility Assays

Two motility assays were tested using the 50-nm diameter beads. First, beads were

attached to the surface and exposed to a motility assay. The second assay bound actin filaments

formed in a solution of ActA-coated 50-nm diameter beads and extract to NEM-myosin surfaces,

similar to the motility assays described in Chapter 4.

5.2.3.1 Attached beads

Aminopropyltriethoxysilane (APES)-treated surfaces were prepared as follows. Glass

coverslips were rinsed in water followed by 100% ethanol. Dry coverslips were then incubated

in a water-free acetone bath for at least 1 hr before transferring to a 2% APES solution in

water-free acetone. Coverslips were incubated in the APES solution for two days, rinsed twice

in acetone and once in water, and set out to dry. These coverslips were incubated in a

1% gluteraldehyde solution for 20 min at room temperature and rinsed twice in distilled water.

The coverslips were dried and then made into flow channels using double-sided tape.

The 50-nm diameter ActA-treated silica beads were diluted in brain extract buffer, added

to the flow channel and incubated for 10 min at room temperature. A 100 mM solution of

glycine methyl ester in brain extract buffer was flowed into to the channel at twice the chamber

volume and incubated for 20 min, followed by brain extract buffer for storage until use. After

incubating on ice for 5 min, supplemented bovine brain extract (see Chapter 4) with either 100%

or 60% extract diluted in brain extract buffer was mixed with 5 [iM of Oregon green 488-labeled

actin and the solution was flowed into the channel. Actin filament structures were visible within









a few minutes. When noted, reactions were fixed with 1% gluteraldehyde after the reaction was

allowed to proceed for 1 to 5 min.

5.2.3.2 NEM-myosin surfaces

Flow-cells with cleaned glass surfaces were treated with 2 [iM NEM-myosin for 1 min

followed by 1% BSA in HS-TBS and 1% BSA in LS-TBS. A motility assay containing a 1 to

200 dilution of ActA-coated silica beads was added to the surfaces. (See Section 4.2.4 for a

description of the motility assay.)

5.2.4 Microscopy and Analysis

All samples were observed using TIRF microscopy. MetaMorph was used to acquire

images and to perform analysis. Fluorescent intensity line-scans were performed on samples to

determine the change in intensity over time at various positions on actin filaments. Rate

constants for the recovery of fluorescence at these positions were calculated by fitting the data to

Equation 5-1.

SEq -ekt
-- --
0 IEq (5-1)

I is the intensity (arbitrary units) at time, t (sec), Io is the initial intensity immediately after

photobleaching, IEq is the equilibrium intensity reached, and k is the rate constant (sec-1).

Microsoft Excel was used to solve for k and IEq by solving for the minimum difference between

the experimental and theoretical values.

5.3 Results

5.3.1 Actin Asters

We observed growth of actin filaments from 50-nm ActA-coated beads covalently bound

to glass coverslips using TIRF. These actin filaments formed actin asters, or star-like structures,

around the beads over time (Figure 5-1). The highest intensity of fluorescence was at the center









of the beads, and the filaments' intensities seemed to decrease with increasing distance from the

bead (Figure 5-2). One explanation of this decrease implies that new monomers were

incorporating at the particle ((+)-end elongation near the bead) and that the older regions of the

filament (the (-)-ends), which have been exposed to light in TIRF and were therefore dimmer due

to photobleaching, were further away from the bead. Another possibility for the decrease in

intensity is that shorter filaments closer to the surface were directly below longer filaments,

which were slightly above the surface. In either case, many of the filaments present

demonstrated the same trend (Figure 5-2).

To investigate monomer addition further, we photobleached fields-of-view by exposing the

samples to the laser at its highest intensity and observing the recovery of the fluorescence.

Figure 5-3 is an example of photobleaching where filaments reappeared on the particle and on

the glass surface in the field-of-view. A closer look indicates that the reappearing filaments on

the surface of the bead seem to correspond to a filament existing on the bead before

photobleaching (Figure 5-4). Not all filaments present before photobleaching reappeared.

The fluorescent intensity of two filaments was plotted against the filament distance from

the center of the bead for four different time points after photobleaching occurred (Figure 5-5).

In the first case, the filament recovered, following the same trend of intensity along the length of

the filament until approximately 1.5 .im away from the particle, where the intensity increased to

above the original intensity. This may be due to a short, free filament landing above the

filament. In the second case, the intensity recovery followed the same trend of intensity along

the length as the original filament, reaching approximately 50% to 75% of the initial intensity.

In both cases, after three minutes of recovery, the fluorescent intensity decreased slightly.









Photobleaching is most likely occurring again after this time period, despite the short exposure

time, causing the fluorescent intensity to decrease.

The fluorescent intensity was also measured as a function of time. We fitted the

fluorescent intensity as a function of time and found both the equilibrium intensity and the rate

constant at various points along the filament length. In Figures 5-6A and B, corresponding to the

filament represented in Figure 5-5A, neither the rate constant nor the equilibrium intensity

followed a clear pattern with respect to the length of the filament. The first two rate constants

may be lower because the points were located near the center of the bead, which did not

photobleach completely.

In Figures 5-6C and D, which correspond to the filament in Figure 5-5B, the rate constant

decreased exponentially over the length of the filament, reaching a final value assumed to

correspond to the background rate of recovery. This implies that recovery near the bead is faster,

i.e., monomers are incorporating in the filament at the bead's surface first. Based on the initial

fluorescent intensity, the region near the bead seems to have a high concentration of monomers,

which could be locally available to the (+)-ends of polymerizing filaments. The equilibrium

intensity remained near the same ratio (compared to the initial intensity before photobleaching)

along the length of the filament, except for a decrease between 0.5 and 1 inm.

5.3.2 Single Actin Filaments

From these results, it is difficult to determine clearly if filaments are incorporating at the

surface of the particle through filament end-tracking motors. Therefore, the brain extract buffer

was diluted to reduce the number of filaments per bead and samples were fixed with

gluteraldehyde to observe a single filament attached to a bead (Figure 5-7). These samples

showed single filaments that were present on particles bound to the surface. The filaments were









all swept in the direction of flow during the fixing step, potentially causing two filaments (on the

same bead) to appear as one.

We next attempted to bind filaments with attached beads to the surface to observe particle

motility. NEM-myosin surfaces bound many filaments, but beads were hard to image from

solution because they were small with large thermal motion (Figure 5-8). An enlarged image of

the modified surfaces shows filaments with a corresponding bead, but it is unclear if they were

attached or simply near each other by chance. The images from this experiment are difficult to

interpret because many filaments may be free filaments in solution that bound to the surface but

are not associated with a bead. Motility assays with and without beads appeared very similar in

TIRF when looking at the fluorescently-labeled F-actin, confirming that F-actin was present in

large concentrations in the extract (Figure 5-9). The actin monomer concentration in the solution

is high (> 5 gM), which provided ideal conditions for the formation of free filaments.

5.4 Discussion

We have attempted to propel small particles by single filament elongation using the

ActA/VASP complex. We found that the fluorescent intensity along the length of the filaments

decreased exponentially from the center of the particle to the end of the filaments, inferring that

the polymerization may be occurring at the bead surface. Fluorescent recovery after

photobleaching showed that, in the case of one filament, faster recovery rates occurred near the

surface of the bead. Other filaments either did not recover or had different intensity trends

compared to the initial filament, leaving the photobleaching results inconclusive. If the

elongation of all filaments was occurring by insertional polymerization, we would expect that the

recovery rate would continue to decrease along the filament length because monomers can only

add to the (+)-end of the filament.









Formation of the actin asters potentially have multiple mechanisms at work that are

difficult to distinguish from one another through fluorescent microscopy. Single actin filaments

are only 7 nm in diameter, making it possible that the free filaments in solution and the

background fluorescence of the surface in TIRF may be distorting some of the observed trends.

Therefore, insertional polymerization may not be solely responsible for the recovery of

fluorescence in our photobleaching experiments. Another explanation of the photobleaching

results could be that multiple filaments are lined up with each other, appearing as one, more

intense filament in certain regions. New filaments may be dropping to the surface as seen in

Figure 5-3 and randomly landing on or around filaments attached to the beads.

In addition, two previously reported studies add to the possible explanations of the

formation of actin stars and growth on beads. Brown, et al. observed actin stars on

polylysine-coated 1-.im diameter polystyrene beads, which can nucleate polar assembly of

filaments. In this case, through myosin subframent Si labeling and observation with an electron

microscope, the filaments were shown to be attached to the bead at the (-)-end and therefore,

polymerized outward by adding monomers to the filament (+)-end (118). Our results hint that

polymerization is occurring at the bead, but we have not conclusively eliminated the possibility

of filaments attached to the particles at their (-)-end by some other mechanism. Vignjevic, et al.

observed star-like actin structures with bundled filaments, not single-filaments, creating the

arms. These stars demonstrated organization of both lamellapodia and filopodia and were

dependent on the concentrations of Arp2/3 and capping protein (119). Through fluorescent

microscopy, we can not distinguish between single filaments and bundles of a few filaments.

Bundling proteins are likely present in the cell extracts, making it possible for bundles of

filaments to form.









Diluting the extract seemed to consistently produce beads with single or a few filaments.

Insight into the mechanism has recently been gained with the use of electron microscopy (EM)

images and TIRF color-change experiments (Sturm, C., J. Phillips, W. Zeile, K. Interliggi, R.

Dickinson, and D. Purich, unpublished). Details of filaments, such as the difference between

bundles and single filaments are hard to resolve in TIRF, therefore, EM was used. Under

specific conditions, a majority of particles attached to filaments only had one filament per

particle. Furthermore, by exchanging the actin from a green fluorescent label to a red fluorescent

label (or vice-versa) in a flow-cell, the color of the labeled actin added second was always

closest to the bead, indicating insertional polymerization (i.e. the (+)-end is attached to the

surface of the bead). In most cases, the filaments grew to an average length of 500 nm for

10 seconds, corresponding to an elongation rate of approximately 3 itm/min (Sturm, C., J.

Phillips, W. Zeile, K. Interliggi, R. Dickinson, and D. Purich, unpublished).

The length may be controlled by the presence of capping protein in the extract and could

possibly be optimized through varying the concentration of capping protein. For the purpose of

control over this system, it may be necessary to use pure component systems which include

actin, Arp2/3, ADF, capping protein, VASP, profilin, and a-actinin (115). A pure component

system would provide control over protein concentrations, and could help to polymerize

filaments for longer time- and length-scales, essentially broadening their application in

nanodevices.

When NEM-myosin surfaces were used, visualization was difficult because many actin

filaments did not correspond to a bead. Due to the extent of nonspecific actin polymerization, it

could not be confirmed that actin filament tails were attached to 50-nm diameter beads when

applied to the NEM-myosin surface. Many filaments are most likely spontaneously forming due









to the high concentration of actin, as indicated by the control sample in which no beads were

added. Solutions to preventing nonspecific filament formation include the use of profilin or

capping proteins.

Insight into the mechanism of actin-based motility suggests that we are making strides

toward single filament elongation that can propel small particles end-tracking motors, such as

formins and possibly ActA/VASP. Single filaments have the advantage over large rocket tails

because they are more flexible, making it easier to manipulate their paths on patterned surfaces.

Motile particles would need to be small enough so they do not effect the fluctuations of the

attached, elongating filament end. The limitation of these experiments is visualization of both

the 50-nm diameter particle and the actin filament in fluorescence at the same time. Biochemical

optimization of the system could lead to better visualization and the development of

single-filament actuators for use in bionanodevices.




























Figure 5-1. Growth of actin filaments/bundles on 50-nm diameter beads. Initially, the 50-nm
beads bound to the surface become surrounded by an actin network. The number of
filaments and, to some extent, the length of the filaments becomes larger and longer
with time. Experiments used full extract strength with an addition of 5 iM of Oregon
green-labeled actin (scale bar = 5 pm).


























B
180
160
140 3
120 -". ', -- 4
d 100

80

60
S40
20


0 1 2 3 4 5
Length (pm)

Figure 5-2. Fluorescent intensity of actin filaments/bundles. A) Enlarged image from Figure
5-1 of 50-nm bead bound to the surface. The fluorescent intensity is greatest in the
center of the bead (scale bar = 3 pm). B) Fluorescent intensity as a function of length
along the filament. The numbers in the legend correspond to the filament labels in A.
A line-scan across the filaments show that for all four cases, the intensity decreased
for approximately 3 pm of length, where it ultimately levels out to the background
intensity. Line-scans began at the beginning of each protrusion at the apparent
surface of the bead.
































Figure 5-3. Actin asters recovery after photobleaching. A) Initial image of asters of actin on
50-nm diameter beads. B) After photobleaching, only a small fluorescent signal is
present at the center of the bead. C) Over time, fluorescence is regained and
filaments reappeared on the 50-nm beads. Filaments not associated with a bead also
reappeared on the surface. Images were taken 1.5 minutes apart (scale bars = 5 im).





























Figure 5-4. Enlarged image of asters reappearing after photobleaching. A) Initial image of
asters of actin on 50-nm beads (from Figure 5-3A). B) After photobleaching, only a
small fluorescent signal is present at the center of the bead. C) Over time,
fluorescence is regained and filaments reappeared on the 50-nm beads. The filaments
that appear during the recovery of photobleaching may correspond to initial filaments
seen in A. Images were taken 1.5 minutes apart (scale bars = 2 gm).











140 B before PB
S-- A PB Inital
120 -AkerPB 1.5 nin

100 -
1>' 100 --AfferPB 3 inn
A fr PB 4.5 in
S80o
S60

40 -
40



0 0.5 1 1.5 2 2.5 3
B

140

120 -

100oo

80
F--I
60

40 -
40
20


0 0.5 1 1.5 2 2.5 3
Length (l)m)

Figure 5-5. Fluorescent intensity ofphotobleached filaments. A) Fluorescent intensity of
filament 1 (Figure 5-4A) as a function of time. At length = 0 tm to 1.5 tim, recovery
with respect to length followed the initial trend. At length greater than 1.5 itm,
fluorescent intensity exceeded the initial intensity and also increased slightly with
increasing length. B) Fluorescent intensity of filament 2 (Figure 5-4B) as a function
of time. The intensity of the filament as a function of length followed the initial
intensity, before photobleaching.












0.63 -

0.58 -

0.53 -

0.48 -

0.43 -

0.38 -


1.2 -

1-

0.8

0.6


. *


S+* #* *


. *


+ +


++*.r **+


Length (pm)


Figure 5-6. Recovery rates and equilibrium intensities of photobleached filaments.
A) Fluorescent recovery rate of filament 1 (Figure 5-4A) as a function of position of
the filament from the center of the bead. B) The ratio of the calculated equilibrium
intensity and the initial intensity before photobleaching of filament 1 (Figure 5-4A).
Note that the intensity recovered between 1.5 and 2 [m is greater than 1,
corresponding to the region of the filament that was brighter than the initial filament.
C) Fluorescent recovery rate of filament 2 (Figure 5-4B). The rates follow an
exponential trend as the length away from the bead increases. D) The ratio of the
calculated equilibrium intensity and the initial intensity of filament 2 (Figure 5-4B).
With the exception of intensities between 0.75 and 1 [im, the equilibrium intensities
to initial intensity ratio seem to be within 0.35 and 0.55.













0.66 -


0.62 -


0.58 -


0.54 -


. .


* ** *


-t II


0.65 -


0.55


0.45 ++ ** *


0.35 -


0.25


+ *


Length (pm)


Figure 5-6. Continued


* #
























Figure 5-7. 50-nm diameter beads bound to surface with single filaments or bundles attached.
Experiments were fixed with gluteraldehyde. Optimization of conditions with the
extract (diluted 4:3 in brain extract buffer) led to a reduced number of filaments per
bead (scale bar = 5 im).



































Figure 5-8. 50-nm diameter beads in solution on NEM-myosin surfaces. A) Instantaneous
image of fluorescent 50-nm diameter particles, which are constantly moving in and
out of the focal plane with time since they are not bound to the surface.
B) Rhodamine-labeled actin filaments bound to NEM-myosin surface in same
field-of-view. C) Overlay of images A and B. The large number of filaments make it
difficult to see any corresponding beads and filaments (scale bar = 10 gm). D)
Enlarged image of beads near filament ends. It is unclear if the beads are attached to
the filaments or if they are near each other (scale bar = 3 gm).


























Figure 5-9. Actin motility assay with and without beads on NEM-myosin surfaces. A) Sample
containing no beads still produced filaments in solution which bind to the
NEM-myosin treated surface. B) Sample containing beads produced and bound
approximately the same amount of filaments as a sample with no beads. Both
samples have 5 .iM of Oregon green-labeled actin (scale bar = 5 .im).









CHAPTER 6
SUMMARY AND FUTURE WORK

Applications that involve the exploitation of biomolecular motors are continuously

expanding to include a broad spectrum from biosensors for single molecule detection to

production of nanoelectromechanical devices. Several methods have already shown promise

towards these types of applications, including the gliding of actin filaments or microtubules on

myosin- or kinesin-modified surfaces, respectively, and the movement of cargo-carrying myosin

or kinesin on immobilized filaments (32). These techniques have yet to explore the potential of

actin polymerization and actin-based motility as possibilities for nanoscale transport.

6.1 Single Actin Filaments

The work presented in Chapter 2 demonstrates a technique that aligns polymerizing actin

filaments on tracks with the potential for transport of material through cargo-carrying myosin or

end-tracking motors bound to filament (+)-ends. Single filaments can potentially attach to

end-tracking motors bound to cargo to propel small nanoparticles in an aligned fashion for

suitable applications that do not change the thermal fluctuations of the filament. Such particles

may include 50-nm diameter particles, antibodies, or quantum dots (2, 4, 14). One important

aspect of this assay is that the density of filaments on the surface can be easily controlled. We

demonstrated that the filament density decreased on surfaces with the addition of profilin to the

actin solution. Higher filament density can be achieved by increasing actin concentration and

actin nuclei, potentially with the addition Arp2/3.

Our experiments showed that elongating actin filaments bound to NEM-myosin tracks

aligned with the track edge in a manner dependent on the track width and NEM-myosin surface

density. We concluded that the alignment of filaments at track boundaries was facilitated by

filament thermal fluctuations, based on the following results. Alignment of actin filaments was









inversely proportional to the track width, with the trend leveling off to slightly above random

alignment at 10-.im wide tracks. This can be explained by the higher ratio of track edges to track

area for the narrower track widths. This increase in ratio causes higher alignment of bound

filaments through more frequent interactions with the track edge. Filaments near track edges

also demonstrated a bias in alignment, regardless of the track width. This is supported by the

conclusion that the probability of an actin filament rebinding once crossing the track edge was

dependent on the angle with which the filament crossed the edge. No filaments elongating

passed the edge at angles greater than 600 realigned.

These results indicate that microcontact printing NEM-myosin is a viable method for the

creation of filament-binding tracks on glass surfaces. One downfall included the diffusion of

NEM-myosin molecules from the tracks to the passivated regions, which most likely occurred

because the NEM-myosin is adsorbed to the surface, not covalently attached (82). Although this

did not seem to affect the final results, some weak binding (to one or a few NEM-myosin

molecules only) occurred in the BSA regions of the surface. Another problem was that

deformations of the stamp occurred, creating tracks of varying sizes when stamped with the

PDMS pattern, as well as some tracks without center regions (120).

The proposed mechanism for the alignment of filaments was supported through

simulations, which used the bending energy of the filament to determine filament shapes and

behavior (108). The simulations predicted the same effects of track width, myosin concentration

and rebinding probability on alignment of filaments as were found experimentally in Chapter 2.

Experimental variations that may account for differences between simulations and experiments

include variations in surface density and filament-binding activity of the substratum. We also

looked at the effect of persistence length on the filaments, finding that the persistence length









does play an important role in the rebinding of the filaments. Because our proposed mechanism

requires bending of filaments, the persistence length was expected to play a key role in the

filament alignment. The simulations will allow for the future exploration of the effect of various

conditions, including different shaped tracks (21).

6.2 Actin-Based Motility

We have reproduced previous methods that use the surface protein, ActA, to induce

actin-based motility by forming actin rocket tails that propel Listeria and particles in vitro.

NEM-myosin was used to confine actin rocket tails to glass surfaces and subsequently, contain

the path of the motile object near the surface without affecting the velocity. The binding of the

actin rocket tails to the surface may be a result of both the surface and the structural features of

the rocket tail. Specifically, the rocket tails contain single actin filaments (or small bundles)

protruding from the sides of the tail, presumably produced by Arp2/3. The NEM-myosin

molecules may bind to these filaments in addition to the exterior filaments in the rocket tail

bundle.

The ability of the surface to contain these large structures and reduce fluctuations in their

paths is a significant step toward the use of the ActA/VASP complex and end-tracking motors in

bionanodevices. Elucidating the mechanism by which binding occurs may help to optimize the

system for applications. We attempted to guide particle propulsion further with the use of

channels on glass surfaces. The channel design requires features such as width, material used

and height to sufficiently prevent the actin rocket tail and attached object from escaping, an

important property for transporting particles in devices. Further optimization of the channel

design is needed to determine the effectiveness of this system for applications.

We expect that this system can be applied to small particles with single filaments attached.

Single filament propulsion is difficult to achieve but extremely useful for bionanodevices. We









have successfully attached ActA-coated beads to surfaces and observed single actin filament

growth from these beads, although the work presented here does not conclusively confirm the

mechanism by which the filaments are attached. We have shown the difficulties in visualizing

and confining single actin filaments to beads in a cell extract on NEM-myosin surfaces.

Difficulties include a high density of filaments (not produced by ActA/VASP) and imaging a

fluctuating bead, both inhibiting the ability to definitively observe a motile bead attached to an

elongating single filament.

6.3 Recommendation for Future Work

6.3.1 Filament-Binding Tracks

The effect of microcontact-printed tracks on the alignment of elongating actin filaments

has been characterized in the presented work. Further work should be aimed towards the

optimization of the mircrocontact printing of NEM-myosin. The technique of patterning proteins

is complicated in that the activity of the protein, which may be dependent on the structure and

position of the protein, must be conserved. Optimization of microcontact printing may provide a

way to ensure the position of NEM-myosin is at its most effective, therefore, increasing the

binding activity and maintaining the integrity of the pattern (20).

Modifications that could be explored include covalently binding NEM-myosin to an

APES-treated surface stamped with gluteraldehyde, ensuring that the myosin heads are exposed.

In addition, it may be beneficial to work with heavy-meromyosin, a smaller, still active part of

the myosin molecule. The smaller molecule may make it easier to maintain activity and

structural integrity, especially if the myosin is covalently bound to a surface (18). Another

possibility is to exploit biotin-streptavidin interactions, which are commonly used in

microcontact printing and easily attached to proteins (18, 24). These techniques may reduce the

diffusion of myosin from the tracks to the passivated regions while making the protein more









effective. Stamp deformation, another problem associated with microcontact printing, may be

reduced by optimizing and standardizing the pressure applied to the stamp during the transfer of

the proteins.

Other work should be focused towards varying the shape of the filament-binding regions to

include curves and corners to determine the effect on the alignment of filaments. In addition,

controlling density of the filaments may help to optimize alignment and elongation. We have

already determined that profilin lowers the density on the surface. Other proteins that may have

an effect on the density or actin network include capping protein, which increases the (+)-end

critical concentration to maintain filament lengths, Arp2/3, which increases nucleation and also

produces branching for networking, and a-actinin, which cross-links filaments into a continuous

network (39). All of these proteins provide advantages to immobilized filament assays by

controlling the characteristics of the filaments on the surface, including the potential for

lengthening the path for myosin-based transport.

6.3.2 Three-Dimensional Surfaces for Larger Structures

Further investigation into the effect of microcontact printing on actin rocket tails

propelling Listeria and larger beads in vitro is recommended. However, it is unlikely that

microcontact printing alone will help to align actin rocket tails, which consist of thousands of

filaments. Three-dimensional devices in combination with NEM-myosin surfaces are expected

to be more apt to guide particle propulsion. These types of devices, with modified surfaces and

topographical features, have been most successful at the guidance of sliding microtubules

compared to systems using only one of the two features (16, 105). Further work on the

optimization of these devices would likely provide the means to guide elongation of actin rocket

tails and their attached particles. The height of the topography, as indicated by our experiments,

is an important parameter to be optimized so that the particles do not climb over the walls, but









rather turn at the walls, following the designated path. Undercuts on the top of the channel walls

may further ensure that beads remain near the surface (17). In addition, devices may need less

exposed glass than those shown in Chapter 4, creating longer channels that span the entire

flow-cell. This design should increase the chances that tails bind to surfaces inside channels.

6.3.2 Use of End-Tracking Motors

To obtain more control over the biochemistry involved in the ActA/VASP and

end-tracking motor systems, it is necessary to test pure component systems, which have

previously been reported to sustain actin-based motility (115). This is particularly important for

the single-filament processive motors, which produced more filaments than just those from the

ActA-coated beads (due to a high actin concentration). Observation of single-filament

elongation is important not only for biodevices, but also for the elucidation of the mechanism by

which ActA/VASP produces force. Although this mechanism is still under debate, it remains

that the force generated can be advantageous for in vitro applications. The Brownian Ratchet

model allows for the release of energy of monomer addition (2 kT) (86). The actoclampin

mechanism allows for the energy released from ATP hydrolysis to also be used, which would

provide 16 kT of accessible energy and a corresponding opposing force of 12 pN (26).

Since formins produce a single, unbranched filament, they provide a simpler system for the

use of end-tracking motors, and should be considered for applications in bionanotechnology. In

previous reports, the stall force and buckling force for single actin filaments were measured by

tethering the filament (+)-ends to formins and attaching another point of the filament to the

surface with NEM-myosin. The buckling force of the filaments is proportional to its stiffness

and inversely proportional to its length. Therefore, the shortest filament formed (0.75 [tm),

produced a force of 1.3 pN (94). The forces from formins, ActA/VASP and other end-tracking

motors can provide many advantageous to micro- and nano-scale applications. In vivo, motors









are efficient and essential to many cellular processes, making them optimal for engineering

future bionanodevices.











APPENDIX
MATLAB CODE

This program simulates the elongation of actin filaments on patterned, filament-binding

surfaces. This program is designed to run multiple filaments in a loop, with one filament

elongating per iteration. We first looked at the effect of the filament probability of rebinding

when leaving the surface by setting the initial position to 4.99 tm with the track edge located at

5 tm. We then varied the number of modes, the binding probability constant, the persistence

length, the final total length of the filaments, and the step size, ds. We also looked at the

alignment of filaments as a function of track width, where the initial position and initial angle of

the filaments were randomly selected. We varied the binding probability constant, the number of

modes, and the persistence length. Lengths and parameters were estimated and varied based on

experimental values.

%This program simulates the elongation of filaments on patterned,
%filament-binding surfaces

clear all

rand('seed', sum(clock)); %Seeds random number generators in MATLAB
randn('seed', sum(clock));

%Number of Runs
ns = 20; %Number of filaments per run
nt = 15*100 %Maximum length per filament Number of steps per filament
nsnt = ns*nt; %Number of maximum possible iterations

%Set Initial Matrices
x=zeros(1,nsnt); %Initial x, y matrices that determine final filament shape
Y=x;
xcross=zeros(1,nsnt); %Initial x, y matrices that determine
ycross=x; %Instantaneous position of elongating filament end

xcross2 = x; %Second to last instantaneous position of filament end
ycross2 = y;
xb=zeros(1,nsnt); %Initial x, y matrices at which binding occurs
yb=xb;
thend = zeros(1,nsnt); %Initial theta matrix at which binding occurs
ibin=xb; %Iteration at which binding occurs
%mostly for troubleshooting purposes
Lbind = xb; %Length of filament binding











ibind=l %Set initial iteration at which binding occurs
k = 1;

%Determine Length of Filament 1
fillen = (15*rand(1,1))+1 %Select random length less than
%15 microns of first filament
nx=round(fillen*100) %Number of steps for initial filament



%Repeat Loop for Each Filament
for count = l:ns %For filament 1 through filament ns
dt=l; %Time increment (seconds)
ds=.01; %Change in length (microns)

%Generate random number matrices for filament count
rnd = rand(l,nx); % Rnd(x) is used to test the probability of binding
rndn=randn(1,nx);

%Input Variables
nmod = 2 % Number of modes
kp=0.01 % Binding probability scaled to kb^-1 (microns^-l*seconds^-l)
width = 8; % Input track width
space = 20; % Input space between tracks
lam = 10; % Persistence length (microns)
v=0.01; %Elongation rate of filaments (microns/sec)

%Randomize initial starting point and theta, initial binding point
x(k) = 0; %Initial x position is 0
y(k)= (width-.01)*rand-(width/2); % Initial y position is randomly chosen
yb(k)=y(k); %Binding occurs in the initial position
xb(k)= x(k); %xb = x(0) and yb = y(0)
thend(k) = (((90*pi/180))*rand); % Direction of filament at time = 0
L = 0; % Length of unbound region initially
xcross(k) = 0; %Binding occurs in the initial position
ycross(k)= y(k); %xcross(0) = x(0) and ycross(0) = y(0)
xcross2(k) = 0;
ycross2(k)= y(k);
initial position = y(k); %Print initial position in command window
initial angle = thend(k); %Print initial angle in command window

%Loop Increases Free Filament Length
for i=k:nx %k = initial iteration of filament
%Filament 1 starts at k =1, nx = filament length
rnds=randn(1,nmod); %Random variable selected for each mode
L=L+v*dt; %Length is increased by ds = v dt

%Determine if Binding Occurs
if rnd(i) < kp*dt*L %Check if binding can occur based on
%Filament length and probability constant
%Equal probibility along length
Lbin = rand(l,l)*L; %Determine random position on
%unbound length that binding occurs

if Lbin > ds %Make sure that Lbin is larger than ds
%program can not continue (matrix can not be formed)











s=0:ds:L; %Create matrix of all positions, s, along free filament
sbind = 0:ds:Lbin; %Create matrix of all positions, sbind,
%along portion of filament that is about to bind

%Calculate maximum amplitudes for each mode
sign = sqrt (2*L./(((0:(nmod-1))+0.5).^2)/lam/pi^2);
modes = sign.*rnds; %Calculate actual amplitudes using
%random numbers less than 1
theta=0*s; %Create initial matrix theta(s)

for im=l:nmod %Calculate theta for every point, s
theta = theta + modes(im)*sin((im-l+.5)*pi*s/L); %theta(1) = 0
%Sum all modes
end

%Determine x and y points of filament from theta and s
xp=zeros(l, length(s)-l); %Set initial matrices for xp and yp
yp=xp;
for j=2:length(s) %j starting at 2 eliminates the point 0,0 from xp
%Trapezoid rule used to integrate
xp(j-l)=(ds/2)*sum((cos(theta(1:(j-1))))+(cos(theta(2:j))));
yp(j-l)=(ds/2)*sum((sin(theta(1:(j-1))))+(sin(theta(2:j))));
end

%Determine positions with rotation of axis rotated
%with respect to the initial theta (binding position of filament)
%x(1) and y(l) are already set to 0
x(ibind+1:ibind+(length(xp)))=x(ibind)+xp*cos(thend(i))-
yp*sin(thend(i));

y(ibind+1:ibind+(length(xp)))=y(ibind)+xp*sin(thend(i))+yp*cos(thend(i));

%Check if binding (Lbind) occurs within track
%Create wave function to allow for the case of multiple tracks
if cos((y(ibind+length(sbind)-l)/((width+space)/2))*pi)-
cos(((width)/(width+space))*pi)>0

%Record filament end positions
ycross(i)=y(ibind + length(xp));
xcross(i)= x(ibind + length(xp));
ycross2(i)=y(ibind-1 + length(xp));
xcross2(i)= x(ibind-1 + length(xp));

ibind=ibind+length(sbind)-1 %Increase ibind (iteration number
%that binding occurs)to new binding position
ibin(i)=ibind;%Create matrix of iterations for trouble-shooting
xb(i)=x(ibind); %Place x, y position of binding in xb and yb
yb(i)=y(ibind);
Lbind(i)= Lbin; %Record the length of filament that binds
%Calculate angle of the filament segment at position of binding
thbind(i)= thend(i)+ theta(length(sbind)-1);
L= L-Lbin %Update length of unbound region
thend(i+1) = thbind(i); %Update angle at which binding occurs
%Free filament will now undulate around











else %If filament binding is NOT within track, no binding occurs
ibind=ibind %Ibind remains the same if no binding occurs
thend(i+l) = thend(i); %Thend remains the same
%(binding angle of last binding spot)
%Record filament end positions
ycross(i)=y(ibind + length(xp));
xcross(i)= x(ibind + length(xp));
ycross2(i)=y(ibind-1 + length(xp));
xcross2(i)= x(ibind-1 + length(xp));
end

else %If Lbind < ds (not enough binding length)
%Repeat calculation of filament shape (Lines 80, 83-97)
s=0:ds:L;
sign = sqrt (2*L./(((0:(nmod-1))+0.5).^2)/lam/pi^2);
modes = sign.*rnds;
theta=0*s;
for im=l:nmod
theta = theta + modes(im)*sin((im-l+.5)*pi*s/L);
end
%Determine x and y points of filament from theta and s
xp=zeros(l,length(s)-l);
yp=xp;
for j=2:length(s)
xp(j-l)=(ds/2)*sum((cos(theta(1:(j-1))))+(cos(theta(2:j))));
yp(j-l)=(ds/2)*sum((sin(theta(1:(j-1))))+(sin(theta(2:j))));
end
% Calculate positions with rotation
x(ibind+1:ibind+(length(xp)))=x(ibind)+xp*cos(thend(i))-
yp*sin(thend(i));

y(ibind+1:ibind+(length(xp)))=y(ibind)+xp*sin(thend(i))+yp*cos(thend(i));

thend(i+1) = thend(i); %Thend remains the same
%Record filament end positions
ycross(i)=y(ibind + length(xp));
xcross(i)= x(ibind + length(xp));
ycross2(i)=y(ibind-1 + length(xp));
xcross2(i)= x(ibind-1 + length(xp));
end

else %If binding probability is less than the random number generator
%Repeat calculation of filament shape (Lines 80, 83-97)
s=0:ds:L;
sign = sqrt (2*L./(((0:(nmod-1))+0.5).^2)/lam/pi^2);
modes = sign.*rnds;
theta=0*s;
for im=l:nmod
theta = theta + modes(im)*sin((im-l+.5)*pi*s/L);
end
%Determine x and y points of filament from theta and s
xp=zeros(1,length(s)-1);
yp=xp;
for j=2:length(s)
xp(j-l)=(ds/2)*sum((cos(theta(1:(j-1))))+(cos(theta(2:j))));
yp(j-l)=(ds/2)*sum((sin(theta(1:(j-1))))+(sin(theta(2:j))));











end
% Calculate positions with rotation
x(ibind+1:ibind+(length(xp)))=x(ibind)+xp*cos(thend(i))-
yp*sin(thend(i));

y(ibind+1:ibind+(length(xp)))=y(ibind)+xp*sin(thend(i))+yp*cos(thend(i));

thend(i+l) = thend(i); %Thend remains the same
%Record filament end positions
ycross(i)=y(ibind + length(xp));
xcross(i)= x(ibind + length(xp));
ycross2(i)=y(ibind-1 + length(xp));
xcross2(i)= x(ibind-1 + length(xp));
end
k = k+1; %Increase k (iteration number for entire simulation

end %End elongation of one filament

ibind = k; %New filament binds at the first iteration of k
fillen = (15*rand(1,1))+1 %Set length of new filament
nx=round(fillen*100) % Set number of iterations for new filament
nx = k + nx; % Increase total number of iterations
end

xprint = x(l:10:nsnt); %Create matrix of final x and y coordinates and theta
yprint = y(1:10:nsnt);
thendi = thend(1:nsnt);

%Create Excel spreadsheets
matrixcross = [thendi', crosss, crosss, xcross2', ycross2',ibin', thbind',
xb', yb', Lbind' ];
matrix2 = [xprint', yprint'];

xlswrite('80ctl C', matrix2, '6'); %Send final positions of filament
xlswrite('80ctl PC', matrixcross, '6'); %Send instantaneous positions of
%filaments and binding information









LIST OF REFERENCES


1. Howard, J. 2001. Mechanics of motor proteins and the cytoskeleton. Sinauer, Sunderland,
MA.

2. Bachand, G. D., S. B. Rivera, A. K. Boal, J. Gaudioso, J. Liu, and B. C. Bunker. 2004.
Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using
microtubules and kinesin motor proteins. Nano Lett. 4:817-821.

3. Bohm, K. J., R. Stracke, P. Muhlig, and E. Unger. 2001. Motor protein-driven
unidirectional transport of micrometer-sized cargoes across isopolar microtubule arrays.
Nanotechnology. 12:238-244.

4. Mansson, A., M. Sundberg, M. Balaz, R. Bunk, I. A. Nicholls, P. Omling, S. Tagerud, and
L. Montelius. 2004. In vitro sliding of actin filaments labelled with single quantum dots.
Biochem. Biophys. Res. Commun. 314:529-534.

5. Muthukrishnan, G., B. M. Hutchins, M. E. Williams, and W. O. Hancock. 2006. Transport
of semiconductor nanocrystals by kinesin molecular motors. Small. 2:626-630.

6. Suda, H., and A. Ishikawa. 1997. Accelerative sliding of myosin-coated glass-beads under
suspended condition from actin paracrystal. Biochem. Biophys. Res. Commun.
237:427-431.

7. Suzuki, N., H. Miyata, S. Ishiwata, and K. Kinosita, Jr. 1996. Preparation of bead-tailed
actin filaments: Estimation of the torque produced by the sliding force in an in vitro
motility assay. Biophys. J. 70:401-408.

8. Bunk, R., J. Klinth, L. Montelius, I. A. Nicholls, P. Omling, S. Tagerud, and A. Mansson.
2003. Actomyosin motility on nanostructured surfaces. Biochem. Biophys. Res. Commun.
301:783-788.

9. Hess, H. 2006. Materials science. Toward devices powered by biomolecular motors.
Science. 312:860-861.

10. Hess, H., G. D. Bachand, and V. Vogel. 2004. Powering nanodevices with biomolecular
motors. Chemistry. 10:2110-2116.

11. Hess, H., J. Clemmens, C. Brunner, R. Doot, S. Luna, K. H. Ernst, and V. Vogel. 2005.
Molecular self-assembly of "nanowires"and "nanospools" using active transport. Nano
Lett. 5:629-633.

12. Jia, L., S. G. Moorjani, T. N. Jackson, and W. O. Hancock. 2004. Microscale transport and
sorting by kinesin molecular motors. Biomed. Microdevices. 6:67-74.

13. Lin, C. T., M. T. Kao, K. Kurabayashi, and E. Meyhofer. 2006. Efficient designs for
powering microscale devices with nanoscale biomolecular motors. Small. 2:281-287.









14. Ramachandran, S., K. H. Ernst, G. D. Bachand, V. Vogel, and H. Hess. 2006. Selective
loading of kinesin-powered molecular shuttles with protein cargo and its application to
biosensing. Small. 2:330-334.

15. Sundberg, M., R. Bunk, N. Albet-Torres, A. Kvennefors, F. Persson, L. Montelius, I. A.
Nicholls, S. Ghatnekar-Nilsson, P. Omling, S. Tagerud, and A. Mansson. 2006. Actin
filament guidance on a chip: Toward high-throughput assays and lab-on-a-chip
applications. Langmuir. 22:7286-7295.

16. Clemmens, J., H. Hess, R. Doot, C. M. Matzke, G. D. Bachand, and V. Vogel. 2004.
Motor-protein "Roundabouts": Microtubules moving on kinesin-coated tracks through
engineered networks. Lab Chip. 4:83-86.

17. Hess, H., C. M. Matzke, R. K. Doot, J. Clemmens, G. D. Bachand, B. C. Bunker, and V.
Vogel. 2003. Molecular shuttles operating undercover: A new photolithographic approach
for the fabrication of structured surfaces supporting directed motility. Nano Lett.
3:1651-1655.

18. Manandhar, P., L. Huang, J. R. Grubich, J. W. Hutchinson, P. B. Chase, and S. Hong.
2005. Highly selective directed assembly of functional actomyosin on Au surfaces.
Langmuir. 21:3213-3216.

19. Nicolau, D. V., H. Suzuki, S. Mashiko, T. Taguchi, and S. Yoshikawa. 1999. Actin motion
on microlithographically functionalized myosin surfaces and tracks. Biophys. J.
77:1126-1134.

20. Sundberg, M., M. Balaz, R. Bunk, J. P. Rosengren-Holmberg, L. Montelius, I. A. Nicholls,
P. Omling, S. Tagerud, and A. Mansson. 2006. Selective spatial localization of actomyosin
motor function by chemical surface patterning. Langmuir. 22:7302-7312.

21. Suzuki, H., A. Yamada, K. Oiwa, H. Nakayama, and S. Mashiko. 1997. Control of actin
moving trajectory by patterned poly(methylmethacrylate) tracks. Biophys. J
72:1997-2001.

22. Reuther, C., L. Hajdo, R. Tucker, A. A. Kasprzak, and S. Diez. 2006. Biotemplated
nanopatterning of planar surfaces with molecular motors. Nano Lett. 6:2177-2183.

23. Asokan, S. B., L. Jawerth, R. L. Carroll, R. E. Cheney, S. Washburn, and R. Superfine.
2003. Two-dimensional manipulation and orientation of actin-myosin systems with
dielectrophoresis. Nano Lett. 3:431-437.

24. Huang, L., P. Manandhar, K. E. Byun, P. B. Chase, and S. Hong. 2006. Selective assembly
and alignment of actin filaments with desired polarity on solid substrates. Langmuir.
22:8635-8638.

25. Dickinson, R. B., L. Caro, and D. L. Purich. 2004. Force generation by cytoskeletal
filament end-tracking proteins. Biophys. J 87:2838-2854.









26. Dickinson, R. B., and D. L. Purich. 2002. Clamped-filament elongation model for
actin-based motors. Biophys. J 82:605-617.

27. Romero, S., C. Le Clainche, D. Didry, C. Egile, D. Pantaloni, and M. F. Carlier. 2004.
Formin is a processive motor that requires profilin to accelerate actin assembly and
associated ATP hydrolysis. Cell. 119:419-429.

28. Dabiri, G. A., J. M. Sanger, D. A. Portnoy, and F. S. Southwick. 1990. Listeria
monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional
actin assembly. Proc. Natl. Acad. Sci. U.S.A. 87:6068-6072.

29. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, movement, and
spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol.
109:1597-1608.

30. Timauer, J. S., J. C. Canman, E. D. Salmon, and T. J. Mitchison. 2002. Ebl targets to
kinetochores with attached, polymerizing microtubules. Mol. Biol. Cell .13:4308-4316.

31. Hess, H., and V. Vogel. 2001. Molecular shuttles made from motor proteins: Active
transport in non-biological environments. Reviews in Molecular Biotechnology. 82:67-85.

32. Bakewell, D. J. G., and D. V. Nicolau. 2007. Protein linear molecular motor-powered
nanodevices. Aust. J. Chem. 60:314-332.

33. Hess, H., J. Clemmens, J. Howard, and V. Vogel. 2002. Surface imaging by self-propelled
nanoscale probes. Nano Lett. 2:113-116.

34. Laki, K., W. J. Bowen, and A. Clark. 1950. The polymerization of proteins; adenosine
triphosphate and the polymerization of actin. J. Gen. Physiol. 33:437-443.

35. Mommaerts, W. F. 1951. Reversible polymerization and ultracentrifugal purification of
actin. J. Biol. Chem. 188:559-565.

36. Otterbein, L. R., P. Graceffa, and R. Dominguez. 2001. The crystal structure of
uncomplexed actin in the ADP state. Science. 293:708-711.

37. Straub, F. B., and G. Feurer. 1950. Adenosinetriphosphate the functional group of actin.
Biochim. Biophys. Acta. 4:455-470.

38. Straub, F. B. 1942. Actin. Stud. Inst. Med. Chem. Univ. Szeged. 2:1-15.

39. Southwick, F. S., and D. L. Purich. 2000. Actin filaments: Self-assembly and regulatory
interactions. In Cellular Microbiology. P. Cossart, P. Boquet, S. Normark, and R.
Rappuoli, editors. ASM Press, Washington, D.C. 153-170.

40. Schafer, D. A., M. D. Welch, L. M. Machesky, P. C. Bridgman, S. M. Meyer, and J. A.
Cooper. 1998. Visualization and molecular analysis of actin assembly in living cells. J.
CellBiol. 143:1919-1930.









41. Wear, M. A., D. A. Schafer, and J. A. Cooper. 2000. Actin dynamics: Assembly and
disassembly of actin networks. Curr. Biol. 10:891-895.

42. Kelleher, J. F., S. J. Atkinson, and T. D. Pollard. 1995. Sequences, structural models, and
cellular localization of the actin-related proteins arp2 and arp3 from acanthamoeba. J. Cell
Biol. 131:385-397.

43. Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2002. Molecular
biology of the cell. Garland Science, New York.

44. Kirschner, M. W. 1980. Implications of treadmilling for the stability and polarity of actin
and tubulin polymers in vivo. J. CellBiol. 86:330-334.

45. Neuhaus, J. M., M. Wanger, T. Keiser, and A. Wegner. 1983. Treadmilling of actin. J.
Muscle Res. Cell. Motil. 4:507-527.

46. Wegner, A. 1976. Head to tail polymerization of actin. J. Mol. Biol. 108:139-150.

47. Kaufmann, S., J. Kas, W. H. Goldmann, E. Sackmann, and G. Isenberg. 1992. Talin
anchors and nucleates actin filaments at lipid membranes. A direct demonstration. FEBS
Lett. 314:203-205.

48. Isambert, H., P. Venier, A. C. Maggs, A. Fattoum, R. Kassab, D. Pantaloni, and M. F.
Carlier. 1995. Flexibility of actin filaments derived from thermal fluctuations. Effect of
bound nucleotide phalloidin and muscle regulatory proteins. J. Biol. Chem.
270:11437-11444.

49. Kas, J., H. Strey, J. X. Tang, D. Finger, R. Ezzell, E. Sackmann, and P. A. Janmey. 1996.
F-actin, a model polymer for semiflexible chains in dilute, semidilute, and liquid
crystalline solutions. Biophys. J. 70:609-625.

50. Sugimoto, Y., M. Tokunaga, Y. Takezawa, M. Ikebe, and K. Wakabayashi. 1995.
Conformational changes of the myosin heads during hydrolysis of ATP as analyzed by
x-ray solution scattering. Biophys. J. 68:29S-33S; discussion 33S-34S.

51. Lymn, R. W., and E. W. Taylor. 1971. Mechanism of adenosine triphosphate hydrolysis by
actomyosin. Biochemistry. 10:4617-4624.

52. Rayment, I., H. M. Holden, M. Whittaker, C. B. Yohn, M. Lorenz, K. C. Holmes, and R.
A. Milligan. 1993. Structure of the actin-myosin complex and its implications for muscle
contraction. Science. 261:58-65.

53. Stossel, T. P. 1993. On the crawling of animal cells. Science. 260:1086-1094.

54. Cassimeris, L., D. Safer, V. T. Nachmias, and S. H. Zigmond. 1992. Thymosin beta 4
sequesters the majority of G-actin in resting human polymorphonuclear leukocytes. J. Cell
Biol. 119:1261-1270.









55. Southwick, F. S., and C. L. Young. 1990. The actin released from profilin-actin complexes
is insufficient to account for the increase in f-actin in chemoattractant-stimulated
polymorphonuclear leukocytes. J. CellBiol. 110:1965-1973.

56. Kang, F., D. L. Purich, and F. S. Southwick. 1999. Profilin promotes barbed-end actin
filament assembly without lowering the critical concentration. J. Biol. Chem.
274:36963-36972.

57. Paavilainen, V. O., E. Bertling, S. Falck, and P. Lappalainen. 2004. Regulation of
cytoskeletal dynamics by actin-monomer-binding proteins. Trends Cell Biol. 14:386-394.

58. Mullins, R. D., J. A. Heuser, and T. D. Pollard. 1998. The interaction of arp2/3 complex
with actin: Nucleation, high affinity pointed end capping, and formation of branching
networks of filaments. Proc. Natl. Acad. Sci. U.S.A. 95:6181-6186.

59. McGough, A. 1998. F-actin-binding proteins. Curr. Opin. Struct. Biol. 8:166-176.

60. Carlier, M. F., V. Laurent, J. Santolini, R. Melki, D. Didry, G. X. Xia, Y. Hong, N. H.
Chua, and D. Pantaloni. 1997. Actin depolymerizing factor (adf/cofilin) enhances the rate
of filament turnover: Implication in actin-based motility. J. CellBiol. 136:1307-1322.

61. Casella, J. F., D. J. Maack, and S. Lin. 1986. Purification and initial characterization of a
protein from skeletal muscle that caps the barbed ends of actin filaments. J. Biol. Chem.
261:10915-10921.

62. Cooper, J. A., and T. D. Pollard. 1982. Methods to measure actin polymerization. Methods
Enzymol. 85:182-211.

63. Blanchoin, L., K. J. Amann, H. N. Higgs, J. B. Marchand, D. A. Kaiser, and T. D. Pollard.
2000. Direct observation of dendritic actin filament networks nucleated by arp2/3 complex
and wasp/scar proteins. Nature. 404:1007-1011.

64. Pollard, T. D., and M. S. Mooseker. 1981. Direct measurement of actin polymerization rate
constants by electron microscopy of actin filaments nucleated by isolated microvillus
cores. J. CellBiol. 88:654-659.

65. Burlacu, S., P. A. Janmey, and J. Borejdo. 1992. Distribution of actin filament lengths
measured by fluorescence microscopy. Am. J. Physiol. 262:C569-577.

66. Xu, J., J. F. Casella, and T. D. Pollard. 1999. Effect of capping protein, capz, on the length
of actin filaments and mechanical properties of actin filament networks. CellMotil.
Cytoskeleton. 42:73-81.

67. Yanagida, T., M. Nakase, K. Nishiyama, and F. Oosawa. 1984. Direct observation of
motion of single F-actin filaments in the presence of myosin. Nature. 307:58-60.

68. Axelrod, D. 2001. Total internal reflection fluorescence microscopy in cell biology.
Traffic. 2:764-774.









69. Amann, K. J., and T. D. Pollard. 2001. Direct real-time observation of actin filament
branching mediated by arp2/3 complex using total internal reflection fluorescence
microscopy. Proc. Natl. Acad. Sci. U.S.A. 98:15009-15013.

70. Kuhn, J. R., and T. D. Pollard. 2005. Real-time measurements of actin filament
polymerization by total internal reflection fluorescence microscopy. Biophys. J
88:1387-1402.

71. Atkinson, M. A., P. K. Lambooy, and E. D. Korn. 1989. Cooperative dependence of the
actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of
filament phosphorylation. J. Biol. Chem. 264:4127-4132.

72. Pemrick, S., and A. Weber. 1976. Mechanism of inhibition of relaxation by
n-ethylmaleimide treatment of myosin. Biochemistry. 15:5193-5198.

73. Amann, K. J., and T. D. Pollard. 2001. The arp2/3 complex nucleates actin filament
branches from the sides of pre-existing filaments. Nat. Cell Biol. 3:306-310.

74. Xia, Y., and G. M. Whitesides. 1998. Soft lithography. AnnualReview ofMaterials
Science. 28:153-184.

75. Kumar, A., and G. M. Whitesides. 1993. Features of gold having micrometer to centimeter
dimensions can be formed through a combination of stamping with an elastomeric stamp
and an alkanethiol "ink" followed by chemical etching. Appl. Phys. Lett. 63:2002-2004.

76. Kind, H., M. Geissler, H. Schmid, B. Michel, K. Kern, and E. Delamarche. 2000. Patterned
electroless deposition of copper by microcontact printing palladium(ii) complexes on
titanium-covered surfaces. Langmuir. 16:6367-6373.

77. Nikitov, S. A., L. Presmanes, P. Tailhades, and D. E. Balabanov. 2002. Magnetic
duplication and contact printing method. Journal ofMagnetism and Magnetic Materials.
241:124-130.

78. Presmanes, L., and P. Tailhades. 2002. Field assisted magnetic contact printing with soft
magnetic patterned thin films. Journal ofMagnetism and Magnetic Materials.
242-245:499-504.

79. Renault, J. P., A. Bernard, A. Bietsch, B. Michel, H. R. Bosshard, E. Delamarche, M.
Krieter, B. Hecht, and U. P. Wild. 2003. Fabricating arrays of single protein molecules on
glass using microcontact printing. J. Phys. Chem. 107:703-711.

80. Xu, L., L. Robert, Q. Ouyang, F. Taddei, Y. Chen, A. B. Lindner, and D. Baigl. 2007.
Microcontact printing of living bacteria arrays with cellular resolution. Nano Lett.
7:2068-2072.

81. Rozkiewicz, D. I., Y. Kraan, M. W. Werten, F. A. de Wolf, V. Subramaniam, B. J. Ravoo,
and D. N. Reinhoudt. 2006. Covalent microcontact printing of proteins for cell patterning.
Chemistry. 12:6290-6297.









82. Quist, A. P., E. Pavlovic, and S. Oscarsson. 2005. Recent advances in microcontact
printing. Anal. Bioanal. Chem. 381:591-600.

83. Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J.
Mitchison. 1994. Involvement of profilin in the actin-based motility of L. monocytogenes
in cells and in cell-free extracts. Cell. 76:505-517.

84. Zeile, W. L., D. L. Purich, and F. S. Southwick. 1996. Recognition of two classes of
oligoproline sequences in profilin-mediated acceleration of actin-based .l/nge//a motility. J.
CellBiol. 133:49-59.

85. Brieher, W. M., M. Coughlin, and T. J. Mitchison. 2004. Fascin-mediated propulsion of
Listeria monocytogenes independent of frequent nucleation by the arp2/3 complex. J. Cell
Biol. 165:233-242.

86. Mogilner, A., and G. Oster. 2003. Force generation by actin polymerization II: The elastic
ratchet and tethered filaments. Biophys. J. 84:1591-1605.

87. Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, and P. Cossart. 1992. L.
monocytogenes-induced actin assembly requires the ActA gene product, a surface protein.
Cell. 68:521-531.

88. Cameron, L. A., P. A. Giardini, F. S. Soo, and J. A. Theriot. 2000. Secrets of actin-based
motility revealed by a bacterial pathogen. Nat. Rev. Mol. Cell Biol. 1:110-119.

89. Cameron, L. A., T. M. Svitkina, D. Vignjevic, J. A. Theriot, and G. G. Borisy. 2001.
Dendritic organization of actin comet tails. Curr. Biol. 11:130-135.

90. Cameron, L. A., M. J. Footer, A. van Oudenaarden, and J. A. Theriot. 1999. Motility of
ActA protein-coated microspheres driven by actin polymerization. Proc. Natl. Acad. Sci.
U.S.A. 96:4908-4913.

91. Kuo, S. C., and J. L. McGrath. 2000. Steps and fluctuations of Listeria monocytogenes
during actin-based motility. Nature. 407:1026-1029.

92. Upadhyaya, A., J. R. Chabot, A. Andreeva, A. Samadani, and A. van Oudenaarden. 2003.
Probing polymerization forces by using actin-propelled lipid vesicles. Proc. Natl. Acad.
Sci. U.S.A. 100:4521-4526.

93. Chang, F., D. Drubin, and P. Nurse. 1997. Cdcl2p, a protein required for cytokinesis in
fission yeast, is a component of the cell division ring and interacts with profilin. J. Cell
Biol. 137:169-182.

94. Kovar, D. R., and T. D. Pollard. 2004. Insertional assembly of actin filament barbed ends
in association with formins produces piconewton forces. Proc. Natl. Acad. Sci. U.S.A.
101:14725-14730.









95. Pruyne, D., M. Evangelista, C. Yang, E. Bi, S. Zigmond, A. Bretscher, and C. Boone.
2002. Role of formins in actin assembly: Nucleation and barbed-end association. Science.
297:612-615.

96. Zigmond, S. H., M. Evangelista, C. Boone, C. Yang, A. C. Dar, F. Sicheri, J. Forkey, and
M. Pring. 2003. Formin leaky cap allows elongation in the presence of tight capping
proteins. Curr. Biol. 13:1820-1823.

97. Mogilner, A., and G. Oster. 1996. Cell motility driven by actin polymerization. Biophys. J.
71:3030-3045.

98. Peskin, C. S., G. M. Odell, and G. F. Oster. 1993. Cellular motions and thermal
fluctuations: The Brownian ratchet. Biophys. J. 65:316-324.

99. Hill, T. L. 1981. Microfilament or microtubule assembly or disassembly against a force.
Proc. Natl. Acad. Sci. U.S.A. 78:5613-5617.

100. Abraham, V. C., V. Krishnamurthi, D. L. Taylor, and F. Lanni. 1999. The actin-based
nanomachine at the leading edge of migrating cells. Biophys. J. 77:1721-1732.

101. Kovar, D. R., J. Q. Wu, and T. D. Pollard. 2005. Profilin-mediated competition between
capping protein and formin cdcl2p during cytokinesis in fission yeast. Mol. Biol. Cell.
16:2313-2324.

102. Romero, S., D. Didry, E. Larquet, N. Boisset, D. Pantaloni, and M. F. Carlier. 2007. How
ATP hydrolysis controls filament assembly from profilin-actin: Implication for formin
processivity. J. Biol. Chem. 282:8435-8445.

103. Kwiatkowski, A. V., F. B. Gertler, and J. J. Loureiro. 2003. Function and regulation of
Ena/VASP proteins. Trends Cell Biol. 13:386-392.

104. Geese, M., K. Schluter, M. Rothkegel, B. M. Jockusch, J. Wehland, and A. S. Sechi. 2000.
Accumulation of profilin II at the surface of Listeria is concomitant with the onset of
motility and correlates with bacterial speed. J. Cell Sci. 113 (Pt 8):1415-1426.

105. Clemmens, J., H. Hess, R. Lipscomb, Y. Hanein, K. F. Bohringer, C. M. Matzke, G. D.
Bachand, B. C. Bunker, and V. Vogel. 2003. Mechanisms of microtubule guiding on
microfabricated kinesin-coated surfaces: Chemical and topographic surface patterns.
Langmuir. 19:10967-10974.

106. Pardee, J. D., and J. A. Spudich. 1982. Purification of muscle actin. Methods Enzymol. 85
Pt B:164-181.

107. Bernard, A., E. Delamarche, H. Schmid, B. Michel, H. R. Bosshard, and H. Biebuyk. 1998.
Printing patterns of proteins. Langmuir. 14:2225-2229.

108. Landau, L. D., and E. M. Lifshitz. 1986. Theory of elasticity. Reed Educational and
Professional Publishing Ltd., New York.









109. Ostap, E. M., T. Yanagida, and D. D. Thomas. 1992. Orientational distribution of
spin-labeled actin oriented by flow. Biophys. J. 63:966-975.

110. Lee, C. S., S. H. Lee, S. S. Park, Y. K. Kim, and B. G. Kim. 2003. Protein patterning on
silicon-based surface using background hydrophobic thin film. Biosens. Bioelectron.
18:437-444.

111. Welch, M. D., J. Rosenblatt, J. Skoble, D. A. Portnoy, and T. J. Mitchison. 1998.
Interaction of human arp2/3 complex and the Listeria monocytogenes ActA protein in actin
filament nucleation. Science. 281:105-108.

112. Plastino, J., I. Lelidis, J. Prost, and C. Sykes. 2004. The effect of diffusion,
depolymerization and nucleation promoting factors on actin gel growth. Eur. Biophys. J
33:310-320.

113. Zeile, W. L., F. Zhang, R. B. Dickinson, and D. L. Purich. 2005. Listeria's right-handed
helical rocket-tail trajectories: Mechanistic implications for force generation in actin-based
motility. CellMotil. Cytoskeleton. 60:121-128.

114. Welch, M. D., A. Iwamatsu, and T. J. Mitchison. 1997. Actin polymerization is induced by
arp2/3 protein complex at the surface ofListeria monocytogenes. Nature. 385:265-269.

115. Loisel, T. P., R. Boujemaa, D. Pantaloni, and M. F. Carlier. 1999. Reconstitution of
actin-based motility of Listeria and .\lnge/ll using pure proteins. Nature. 401:613-616.

116. Cameron, L. A., J. R. Robbins, M. J. Footer, and J. A. Theriot. 2004. Biophysical
parameters influence actin-based movement, trajectory, and initiation in a cell-free system.
Mol. Biol. Cell. 15:2312-2323.

117. Oudenaarden, A., and J. A. Theriot. 1999. Cooperative symmetry-breaking by actin
polymerization in a model for cell motility. Nat. CellBiol. 1:493-499.

118. Brown, S. S., and J. A. Spudich. 1979. Nucleation of polar actin filament assembly by a
positively charged surface. J. CellBiol. 80:499-504.

119. Vignjevic, D., D. Yarar, M. D. Welch, J. Peloquin, T. Svitkina, and G. G. Borisy. 2003.
Formation of filopodia-like bundles in vitro from a dendritic network. J. Cell Biol.
160:951-962.

120. Rogers, J. A., K. E. Paul, and G. M. Whitesides. 1998. Quantifying distortions in soft
lithography. Journal of Vacuum Science and Technology B. 16:88-97.









BIOGRAPHICAL SKETCH

Kimberly A. Interliggi was born and raised in Canton, Ohio. She graduated from Jackson

High School in 2000 and attended Ohio University on a full-tuition scholarship. She received

her BSChe Magnaa cum laude) in June 2003. In August 2003, she enrolled in the PhD program

in the Department of Chemical Engineering at the University of Florida. She began working for

Dr. Richard Dickinson in January 2004 and received her doctorate in December 2007.





PAGE 1

1 CHARACTERIZATION OF ACTIN-BASED MO TILITY ON MODIFIED SURFACES FOR IN VITRO APPLICATIONS IN NANODEVIC ES By KIMBERLY A. INTERLIGGI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Kimberly A. Interliggi

PAGE 3

3 To my encouraging parents, Denni and Vicki Interliggi, my siblings, Jen and Tom, and my boyfriend, Matt Crim, for their continued support.

PAGE 4

4 ACKNOWLEDGMENTS I thank m y advisor, Dr. Richard B. Dickinson, fo r his support over the past four years. His guidance and encouragement have helped me succeed in many aspects of my graduate experience. I appreciate Dr. Yiider Tseng for al ways expressing his interest in my research and career and encouraging me along the way. I thank Dr. Spyros Svoronos for his dedication as a committee member. I acknowledge Dr. Anuj Chauhan for his guidance and helpful advice from the very beginning of my graduate career. I also thank Dr. Jennifer Curtis for her dedication to the graduate students in this department. Fi nally, I acknowledge the faculty and staff in the Department of Chemical Engineering for continuously supporting my efforts in many aspects of my graduate school career. I am grateful to have had the opportunity to wo rk with Dr. Daniel Purich in the Department of Biochemistry. Dr. Purich was involved on a da ily basis with my lab work and was constantly teaching me new skills and sharing new ideas wi th me. I also thank Dr. William Zeile, who taught me many experimental and research techniques, and was always willing to listen to my research problems and help me work through th em. Dr. Joseph Phillips and Dr. Fangliang Zhang are greatly appreciated for their companionship and knowledge in the lab. I also thank Dr. Suzanne Ciftan-Hens and Dr. Gary McGuire at In ternational Technology Center in Raleigh, NC for their collaboration on this project. I thank Dr. Adam Feinberg, who was instrumental in helping me start my project. I thank the members of my group, who were consistent ly helping me to understand and work through daily problems and always provided good compa ny: Dr. Luzelena Caro, Dr. Colin Sturm, Gaurav Misra, Dr. Jeff Sharp, Dr. Huilian Ma, and Adam Wulkan. I also thank Andre Baran for his assistance and c onstant support.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES.........................................................................................................................8 LIST OF ABBREVIATIONS........................................................................................................ 11 ABSTRACT...................................................................................................................................12 CHAP TER 1 INTRODUCTION..................................................................................................................14 1.1 Actin..............................................................................................................................15 1.1.1 Filament Growth............................................................................................... 15 1.1.2 Actomyosin....................................................................................................... 16 1.1.3 Actin-Associated Proteins.................................................................................17 1.2 Experimental Methods for Actin Polym erization on Modified Substrata.................... 18 1.2.1 Total Internal Reflectio n Fluorescence Microscopy ......................................... 18 1.2.2 Microcontact Printing....................................................................................... 21 1.3 Actin-Based Motility..................................................................................................... 22 1.3.1 Listeria and Particle Motility ............................................................................ 22 1.3.2 Mechanisms...................................................................................................... 25 1.4 Bionanodevices.............................................................................................................26 1.4.1 Sliding Filament Assay.....................................................................................27 1.4.2 Immobilized Filament Assay............................................................................28 1.4.3 Material Transport.............................................................................................29 2 GUIDANCE OF ACTIN FILAMENT ELO NGATION ON FILAMENT -BINDING TRACKS......................................................................................................................... ........32 2.1 Introduction.............................................................................................................. .....32 2.2 Materials and Methods..................................................................................................32 2.2.1 Protein Preparation............................................................................................32 2.2.2 Microcontact Printing....................................................................................... 34 2.2.3 Actin Polymerization........................................................................................ 35 2.2.4 TIRF Microscopy and Data Analysis................................................................ 36 2.3 Results................................................................................................................... ........38 2.3.1 Filament Binding to Stamped Surfaces............................................................. 38 2.3.2 Alignment of Filaments..................................................................................... 39 2.3.3 NEM-Myosin Concentration.............................................................................41 2.3.4 Control of Actin Polymerization....................................................................... 42 2.4 Discussion.....................................................................................................................43 2.4.1 Mechanism for Filament Alignment................................................................. 43 2.4.2 Potential and Applications................................................................................45

PAGE 6

6 3 SIMULATING ACTIN FILAMENT ELON GATION ON M ODIFIED SURFACES......... 63 3.1 Introduction.............................................................................................................. .....63 3.2 Methodology.................................................................................................................63 3.3 Results................................................................................................................... ........66 3.3.1 Description of Simulated Filament Elongation.................................................66 3.3.2 Probability of Filament Rebinding....................................................................67 3.3.3 Alignment of Filaments..................................................................................... 70 3.4 Discussion.....................................................................................................................71 4 ACTIN-BASED MOTILITY OF LISTER IA AND P ARTICLES ON MODIFIED SURFACES....................................................................................................................... .....86 4.1 Introduction.............................................................................................................. .....86 4.2 Materials and Methods..................................................................................................86 4.2.1 Listeria monocytogenes Growth and Protein Purification ................................ 86 4.2.2 Bead Preparation............................................................................................... 88 4.2.3 Motility Assay...................................................................................................89 4.2.4 Fabrication of Channel Devices........................................................................ 89 4.2.5 Microscopy and Analysis.................................................................................. 90 4.3 Results................................................................................................................... ........91 4.3.1 Confining Particle Propulsion to the Surface.................................................... 91 4.3.2 Effectiveness of NEM-Myosin Surfaces...........................................................92 4.3.3 Particle Velocity and Tail Characterization...................................................... 93 4.3.4 Guiding Particle Propulsion..............................................................................95 4.4 Discussion.....................................................................................................................96 4.4.1 Mechanics of Actin Rocket Tails on Surfaces.................................................. 96 4.4.2 Biochemical Considerations............................................................................. 97 4.4.3 Considerations for Bionanotechnology.............................................................98 5 SINGLE FILAMENT ACTIN-BASED MOTI LITY OF PARTICLES..............................113 5.1 Introduction.............................................................................................................. ...113 5.2 Materials and Methods................................................................................................114 5.2.1 Protein Preparations........................................................................................114 5.2.2 Bead Functionalization................................................................................... 114 5.2.3 Motility Assays...............................................................................................115 5.2.3.1 Attached beads..................................................................................115 5.2.3.2 NEM-myosin surfaces...................................................................... 116 5.2.4 Microscopy and Analysis................................................................................ 116 5.3 Results................................................................................................................... ......116 5.3.1 Actin Asters.....................................................................................................116 5.3.2 Single Actin Filaments....................................................................................118 5.4 Discussion...................................................................................................................119

PAGE 7

7 6 SUMMARY AND FUTURE WORK.................................................................................. 133 6.1 Single Actin Filaments................................................................................................133 6.2 Actin-Based Motility................................................................................................... 135 6.3 Recommendation for Future Work............................................................................. 136 6.3.1 Filament-Binding Tracks................................................................................ 136 6.3.2 Three-Dimensional Surfaces for Larger Structures........................................ 137 6.3.2 Use of End-Tracking Motors.......................................................................... 138 APPENDIX: MATLAB CODE................................................................................................... 140 LIST OF REFERENCES.............................................................................................................145 BIOGRAPHICAL SKETCH.......................................................................................................154

PAGE 8

8 LIST OF FIGURES Figure page 1-1 Treadmilling of an actin filament...................................................................................... 30 1-2 Experimental set-up for binding filaments to a surface..................................................... 30 1-3 Schematic of microcont act printing protein on glass. ........................................................31 2-1 Images of microcontact-printe d m yosin tracks on a glass coverslip................................. 47 2-2 Time-lapse image of actin filaments on a BSA-stamped surface...................................... 48 2-3 Total internal reflection fluorescen ce m icroscopy images of elongating actin filaments on NEM-myosin tracks...................................................................................... 49 2-4 Filaments in BSA region undergo large thermal undulations............................................ 49 2-5 Total internal reflec tion fluorescence microscopy imag es of undulating ends of elongating filaments...........................................................................................................50 2-6 Filament elongating pass ed the track edge at sm all ......................................................50 2-7 Filament alignment as a function of filament density on tracks........................................ 51 2-8 Effect of track wi dth on filam ent alignment...................................................................... 52 2-9 Histogram showing the fraction, f of filament ends that rebind to tracks after their elongating ends cross track boundaries.............................................................................. 54 2-10 Scatter plot showing each segment alignm ent with the track edge as a function of the distance from the track edge .............................................................................................. 55 2-11 Dependence of filament alignment a nd elongation rate on the concentration of NEMmyosin......................................................................................................................56 2-12 Scatter plots showing the alignm ent of individual filam ent segments.............................. 57 2-13 Filaments accumulate in the BSA region of the stamped surface..................................... 59 2-14 Incubation time of lyophilized rhodamine actin................................................................ 59 2-15 Increasing the concentration of profil in visibly decreases the density of actin filam ents on NEM-myosin treated surfaces....................................................................... 60 2-16 Elongation rate of actin filaments as a function of profilin con centration........................ 61

PAGE 9

9 2-17 Illustration of the likely mechanism for actin filam ent alignment on NEM-myosin tracks......................................................................................................................... .........62 3-1 The xand y-positions of elongating filam ent ends as a function of time......................... 75 3-2 Effect of change in length (stepsize of si mulation) on the filament rebinding probability.................................................................................................................... ......77 3-3 Effect of the number of modes on filam ent rebinding probability.................................... 78 3-4 Effect of total filament length on filam ent rebinding probability...................................... 79 3-5 Effect of binding probability constant, Kp (m-1sec-1), on filament rebinding probability.................................................................................................................... ......80 3-6 Effect of persistence length on filament rebinding probability. ........................................81 3-7 Effect of track width and number of m odes on the alignment of filaments...................... 82 3-8 Effect of track width and binding probability constant on the a lignm ent of filaments..... 83 3-9 Effect of binding probability on the alignm ent of filaments.............................................. 84 3-10 Effect of track width and persiste nce length on the alignm ent of filaments...................... 85 4-1 Listeria and 500-nm diameter bead prope lled by actin rocket tails................................. 100 4-2 Rotation of a helical actin rocket tail in solution ............................................................. 100 4-3 Listeria rocket tails on NEMmyosin and BSA-treated surfaces.....................................101 4-4 500-nm diameter beads attached to rocket tails bound on NEM-m yosin and BSA-treated surfaces....................................................................................................... 101 4-5 Fields-of-view with large percentage of tails bound to surface ....................................... 102 4-6 Fraction of actin rocket tails bound to NEMmyosin and BSA-treated surfaces............ 103 4-7 Change in xand y-position over time for beads on NEMmyosin and BSA surfaces... 104 4-8 Helical actin rocket tail conf ined to NEM-m yosin-treated surfaces................................ 104 4-9 Actin rocket tails in total internal reflection fluorescence m icroscopy........................... 105 4-10 Magnified image of actin rocket tail with protruding filam ents...................................... 106 4-11 Actin tail elongation on NEM-myosin and BSA surfaces............................................... 107 4-12 Average tail elongation rates determined from the slope of a best-fit line...................... 108

PAGE 10

10 4-13 NEM-myosin surface effect on persistence of tail ........................................................... 109 4-14 Stamped surfaces with 500-nm di a meter beads attached to actin tails............................ 110 4-15 Actin tails bound to NEM-myosin-treat ed exposed glass of fabricated device. ..............111 4-16 Actin rocket tail encounters CYTOP wall....................................................................... 112 5-1 Growth of actin filaments/ bundles on 50-nm diameter beads......................................... 123 5-2 Fluorescent intensity of actin filam ents/bundles.............................................................. 124 5-3 Actin asters rec overy after photobleaching......................................................................125 5-4 Enlarged image of asters reappearing after photobleaching............................................ 126 5-5 Fluorescent intensity of photobleached filam ents............................................................127 5-6 Recovery rates and equilibrium intensities of photobleached filam ents......................... 128 5-7 50-nm diameter beads bound to surface with single filaments or bundles...................... 130 5-8 50-nm diameter beads in solution on NEM-myosin surfaces.......................................... 131 5-9 Actin motility assay with and without beads on NEM-myosin surfaces......................... 132

PAGE 11

11 LIST OF ABBREVIATIONS ADF Actin depolymerizing factor ADP Adenosine diphosphate AFM Atomic force microscopy APES 3-aminopropyltriethoxysilane Arp2/3 Actin related proteins 2/3 ATP Adenosine triphosphate BHI Brain-heart infusion media BS3 Bis(sulfosuccinimidyl suberate) BSA Bovine serum albumin DMSO Dimethyl sulfoxide DTT Dithiothreitol EDTA Ethylenediamine tetraacetic acid EGTA Ethylene glycol tetraacetic acid EM Electron microscopy HMM Heavy meromyosin NEM N-ethylmaleimide PDMS Polydimethylsiloxane PMSF Phenylmethanesulphonyl fluoride TIRF Total internal reflection fluorescence VASP Vasodilator-stimulated phosphoprotein

PAGE 12

12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF ACTIN-BASED MO TILITY ON MODIFIED SURFACES FOR IN VITRO APPLICATIONS IN NANODEVIC ES By Kimberly A. Interliggi December 2007 Chair: Richard Dickinson Major: Chemical Engineering The cytoskeletal protein actin generates for ces for various processe s by polymerizing into filaments. In vivo actin works with the motor protein m yosin to produce muscle contractions and with proteins acting as end-tracking motors responsible for cell and bacterial motility, such as the motility of Listeria monocytogenes End-tracking proteins bi nd the polymerizing end of an actin filament to a motile su rface, creating persiste nt attachment during filament elongation. Both types of motors use the energy from ATP hydrolysis and can be exploited in vitro in bionanodevices, which require forces to transpor t objects on a microor nano-scale, possibly against flow or diffusion gradients. Our study has focused on the guidance of singl e-filament elongation and filament bundles (rocket tails) to orient elongation in vitro Microcontact printing, a tech nique that stamps protein patterns onto glass surfaces through adsorption, was used to create filament-binding tracks of modified myosin (void of its motor activity), which successf ully bound and guided single actin filament elongation in a manner dependent on track width and surface conditions. These results confirm the capability of this method to be used for the motility of obj ects attached to single actin filaments and for the creation of immobilized tracks of actin filaments for myosin-mediated

PAGE 13

13 transport. Simulations were used to characterize the system further and have the ability to help make predictions for other types of filaments and systems. Modified myosin surfaces also confined actin rocket tails attached to particles and bacteria, reducing the Brownian motion of the motile objects. Channels formed through photolithographic techniques on glass surfaces were us ed to attempt to guide these particles. Single actin filaments attached to smaller partic les were also characte rized to determine the potential for single-filament propulsion in nanodev ices. We conclude that actin filament-binding proteins can be applied to surfaces using adso rption and microcontact printing and that this technique is effective in bindi ng and guiding filaments in various systems, including single and bundled filaments. We predic t this technique can be applied to other systems undergoing actin-based motility, making it a vers atile method for bionanotechnology.

PAGE 14

14 CHAPTER 1 INTRODUCTION Muscle con traction, cell movement intracellular bacteria motili ty and cell division are all processes that involve biomolecular motors, a group of protei ns that utilize the energy from adenosine triphosphate (ATP) hydrolysis to do work on a system (1). These biomolecular motors can be exploited for the in vitro transport of nano-cargo (e.g. beads, bacteria, DNA) (2-7), as well as in biosensors, microfluidic devices and microand nanoelectromechanical systems (MEMS and NEMS) (8-15). One approach ha s been to pattern surface regions with a high-affinity for the adsorption of the molecular mo tors kinesin and myosin to guide transport of pre-assembled microtubules and actin filaments, respectively (8, 15-21). Other methods rely on surface-immobilized microtubules and actin filaments to create tracks for kinesin-mediated (3, 5, 22) and myosin-mediated particle transport (6, 23, 24). Current research has been aimed toward unde rstanding another type of biomolecular motor that couples force-producing, polymerizing filaments to a motile object (25-27). These motors, known as filament end-tracking proteins, are t hought to take part in the formation of lamellapodia and filopodia during cell craw ling, propulsion of bacteria, such as Listeria monocytogenes (28, 29), and chromosome separation dur ing cell division (30). End-tracking motors provide a versatile mechanism for force generation in vitro including the potential to be used as microand nano-scale actuators that can transport objects against obstacles, including opposing forces. Both myosin and end-tracking motors provi de many advantages to bionanotechnology. Nanofluidics is one method for high-throughput anal ysis of biological material, but due to the high surface to volume ratio, friction can cause si gnificant losses. Molecular motor systems can transport material without flow, therefore elimina ting friction losses (10, 31). Other applications

PAGE 15

15 of biomolecular motors in nanodevices are broad and include biomaterials that no longer depend on equilibrium conditions for effectiveness (10) extremely sensitive and fast single-molecule detection (32), and nano-scale information storage and processing through surface-probing (33). Both surface modifications and bi ochemical parameters play a key role in the exploitation of biomolecular motors. We have focused on using suitably patterned surfaces to place and guide elongating actin filaments or bundles of filaments a ttached to particles. 1.1 Actin Globular actin (G-actin), a 43,000 Da protein w ith a radius of 2.7 nm is a cytoskeletal protein found in eukaryotic cells. It has a d eep cleft for binding ATP or adenosine diphosphate (ADP) and an associated cation (Ca2+ or Mg2+) (34-37). G-actin can pol ymerize into filaments (F-actin) under conditions of high ionic strength in vitro producing a 7-nm diameter semi-flexible, double-helical filament with repeats every 37 nm (1, 38). Within the filament, actin monomers align with all of their clefts poin ting in the same direction (toward the (-)-end). This asymmetrical orientation causes polarity in the filament, with a faster-polymerizing (+)-end and a slower polymerizing (-)-end. 1.1.1 Filament Growth Actin polymerization includes a nucleation step, followed by filament elongation and treadmilling (39). Nucleation requires that three ATP-actin monomers come together to form a polymerization nuclei. In vivo and in cell extract systems, ac tin-related protein 2/3 (Arp2/3) complex has been experimentally confirmed to be located at the site of actin filament assembly and assumed to serve as a polymerization nuclei ( 40, 41). As its name implies, Arp2/3 has two subunits with similar structures to actin (42). Elongation occurs when ATP-actin monomers add to the filament (+)-end, followed by hydrolysis of the monomer-bound ATP molecule during or slightly after the addition of the next monomer, creating ADP-Pi-actin monomers within the

PAGE 16

16 filament. The Pi is released, forming ADP-actin mono mers near the (-)-end. The hydrolysis results in a (+)-end rich in ATP-actin a nd a (-)-end rich in ADP-actin (36). In vitro the (+)-end has a lower critical concentrati on than the (-)-end (0.1 and 0.6 M respectively (43)), and when the concentration of free G-actin in solution is between these two values, a behavior known as treadmilling occurs (Figure 1-1) During treadmilling, the filament polymerizes at the (+)-end by the addition of ATP-G-actin and depolymerizes at the (-)-end. Once free from the filament (-)-end, ADP-actin releases its nucleotide and binds to ATP, replenishing the monomer pool for (+)-end elongation and starti ng the cycle over (44-46). In vitro solutions of F-actin have been found to c ontain filaments with lengths of tens of microns (47). Filaments undergo Brownian moti on in solution, which can be modeled based on filament length, L and persistence length, which is the characteristic length over which thermal undulations cause the filament orientation to be uncorrelated. For actin, has been found to be between approximately 10 and 20 m, depending on the conditions (48, 49). Because and L are on the same length scale, F-actin is considered to be a semi-flexible polymer. The thermal motion of phalloidin-sta bilized filaments has been quantitatively described by using the bending energy of a rod w ith both ends free and then performing a normal mode analysis of the bending excitations (49). 1.1.2 Actomyosin The actin/myosin complex (actomyosin) is a type of biomolecular motor that is mainly responsible for the contraction of muscles but also plays a role in actin filament dynamics in the cytoskeleton. Myosin, a 500 kDa dimeric protein, has two heads that interact with an actin filament and through the hydrolysis of myos in-bound ATP, move along the length of the filament. The hydrolysis of ATP to ADP causes the entire head region to bend in a hinge-like

PAGE 17

17 manner due to local conformational changes near the nucleotide binding s ite (50). ATP first binds to myosin attached to an actin filame nt, causing the actin filament to immediately dissociate. The ATP hydrolyzes to form ADP-bound myosin, changing the structure of the myosin head from bent to extended and allowing fo r the myosin to rebind to the actin filament. The ADP is released, causing the myosin head to bend at the hinge (known as a power stroke), moving the actin filament 110 (51, 52). Myosin then binds to a new ATP molecule and the cycle repeats. 1.1.3 Actin-Associated Proteins The cytoplasm of cells contains approximately a concentration of actin 600 to 1200 times the (+)-end critical concen tration of purified actin. Therefore, the cell uses various actin-binding proteins to regulate actin polym erization and depolymerization (53). Two of these proteins include thymosin4 (5 kDa) and profilin (15 kDa), which both bind with higher affinity to ATP-G-actin than ADP-G-actin (54, 55). Thymosin4 sequesters actin monomers and prevents them from adding to filament (+)-ends while profilin aids in polymerization by catalyzing the exchange of ADP to ATP on G-actin and ushering ATP-G-actin to filament (+)-ends (56). Profilin also prevents G-actin from forming nuclei, which can control the density of filaments (57). Arp2/3 complex, as mentioned previously, increa ses the nucleation rate of filaments. In addition, it plays a key role in the development of actin networks. Arps are responsible for branching of filaments by binding to the side of actin filaments or filament (-)-ends to nucleate daughter filaments that extend from the mother f ilament at 70 (58). Another important group of proteins is actin cross-linking proteins, such as -actinin, which help to create various actin structures and networks by connecting fila ments (59). Actin depolymerizing factor (ADF)/cofilins (15-20 kDa) bind with high affi nity to ADP-F-actin, causing an acceleration of

PAGE 18

18 the depolymerization of filaments from the (-)-ends and an increase of the free monomer concentration (60). Capping proteins are also pr esent in cells to cap filament ends, inhibiting elongation (61). These proteins regulate the dynamics of the actin network by controlling the length of filaments, preventing an excess of fila ments, and ensuring G-actin concentrations are sufficient for various cellular activities. 1.2 Experimental Methods for Actin Po lymeriz ation on Modified Substrata Several previous experimental methods have be en used to measure the extent of F-actin elongation and calculate the elongation rate in vitro These include cap illary viscometry, difference spectra, and fluorescence with pyren e-labeled actin monomers (62). The major drawbacks of these methods are that filament lengths can not always be determined, and elongation can be monitored, but not visualized, in real-time. Si nce single actin filaments are 7 nm in diameter, the only ways to observe th ese filaments are through fluorescent labeling or electron microscopy (63, 64). Previous experime nts used phalloidin, a toxin that binds to and fixes actin filaments to prevent them from depol ymerizing, in order to measure the length and structure of actin filaments w ith fluorescent microscopy (63, 6567). Recently, polymerization assays have been enhanced by total internal reflection fluorescence (TIRF) microscopy, which allows for real-time visualiza tion of elongating actin filament s bound to or near a surface. Manipulation of the substrata th rough microcontact printing, a tech nique used for a wide variety of applications, includ ing confinement of cell growth and proteins to a specific pattern, can provide spatial control over filament binding to surfaces. 1.2.1 Total Internal Reflecti on Fluorescence Microscopy Until recently, fluorescently-labeled phalloidin was the best method for visualization of filaments in fluorescence because fluorescently-lab eling actin monomers directly caused a high background signal from the solution, flooding out the resolution of filaments. Because both EM

PAGE 19

19 and phalloidin-labeling fix the filaments at a static point, these techniques still prevent visualization of filament elongation in real-time. The development of TIRF microscopy allows for filaments bound to surfaces to be visualized in a solution of fluoresce ntly-labeled monomers by only illuminating the 200 nm near the surface of the sample, thus minimizing background fluorescence. Therefore, any fl uorophores within the bulk of the sample are not excited, and signals from the excited fluorophores that do em it light are not obscured by any out-of-focus fluorophores (68). The basis of TIRF microscopy is the evanescen t wave created when the light is totally internally reflected at an in terface between two mediums with different refractive indices. Specifically, the light travels from a medium w ith a high refractive inde x to one with a low refractive index. The evanescent wave is created wh en light is reflected at an angle equal to or lower than the critical angle, c, and has the same frequency as the incident light but propagates parallel to the interface (Figure 1-2). The critical angle is cal culated from Snells law (Equation 1-1) and is dependent on the refractive indi ces of the two mediums at the interface. n1 sin 1 = n2 sin 2 (1-1) The refractive index of the aqueous buffer phase is n2, and n1 is the refractive index of the glass phase. At the critical angle, re fraction occurs at an angle of 90 ; therefore sin 2 = 1, and the critical angle is defi ned by Equation 1-2. sin c = n2/n1 1 (1-2) Typically, a laser beam travel s through a glass medium firs t (n = 1.52). Contacting this layer is an aqueous buffer phase containing the specimen to be observed. This solution phase has a lower refractive index (n = 1.33-1.37 for typical aqueous medi ums) than the solid phase.

PAGE 20

20 The intensity of the evanescent wave decreases exponentially from the surface to approximately 200 nm, at which point it di sappears (Equation 1-3). d z0IzI e (1-3) The intensity at any position, I(z) is exponentia lly decreased from the initial intensity I(0) by a function of the ratio of the position from th e interface and the penetration depth, d (Equation 1-4). 2 1. 2 2 22 1 on sinn 4 d (1-4) The penetration depth is dependent on the refractive indices, the angle of incidence, and the wavelength of the incident light in a vacuum, o (68). Actin filaments containing fluorescently-lab eled monomers bound to a surface are clearly resolved in TIRF. One way to attach f ilaments is by adsorbing N-ethylmaleimide (NEM)-myosin to glass (69, 70). NEM binds to thiol groups on myosin, irreversibly inactivating the catalytic site for ATP hydrolysis (71, 72). This allows an actin filament to remain attached to the myosin molecule (Figure 1-2). Amann and Pollard directly observe d individual filaments formed from a solution of G-actin partially labeled with rhodamine by using NEM-myosin to bind the filaments to the surface. Their experime nts confirmed the elongation rate constants of the (+)-ends of actin f ilaments as previously found by EM. They also determined that rhodamine-labeled actin was a kinetically inac tive label, making it us eful for visualizing elongating filaments (73). They showed that Ar p2/3 branching in solution occurred at random sites along the filament length, confirming previously published results that used phalloidin stabilization (73). Kuhn and Pollard studied both polymerization and depolymerization rates

PAGE 21

21 further using TIRF to visualize filament (+)an d (-)-end elongation in real-time under a variety of conditions (70). 1.2.2 Microcontact Printing In our experiments, we patterned NEM-myos in on glass coverslips using microcontact printing to bind actin filaments to the su rface and guide their elongation (Figure 1-3). Microcontact printing is part of a group of techniques known as soft lithography, which uses a patterned elastomer as a stamp, mold, or mask in replacement of a rigid photomask associated with photolithography to create micropatterns (7 4). Polydimethylsiloxane (PDMS) elastomers are most commonly used for microcontact printing because of a number of advantages, including chemical and thermal stability and durability. This technique was initially used to form self-assembled monolayers (SAMS), or monolay ers that form due to the spontaneous aggregation and organization of molecules into a stable structure without the use of covalent bonds (74). Kumar and Whitesides first demonstr ated this technique by inking patterned PDMS with feature sizes 1 to 100 m with hexadecanethiol and then stamping it onto a gold surface (75). The applications of microcontact-printed SAMS were furthered by the selective deposition of other materials onto the monolayers, creatin g the same pattern with a different exposed material. These materials included conducting po lymers, inorganic salts, metals, and proteins (74). Microcontact printing has more recently expa nded to transfer metals, electromagnetic particles, electrochemical particles, and proteins directly. Kind, et al. de monstrated the inking of a solution of Palladium(II) onto a Titanium-coated silicon wafer (76). Iron dots patterned onto a magnetic slave-film have successfully duplicated magnetic signals (77, 78). Applications have also moved toward biology, with Renault, et al. showing that microcontact printing fluorescently-labeled antibodies co uld provide a method for studying single protein molecules on

PAGE 22

22 surfaces (79). Xu, et al. used a PD MS stamp to transfer patterns of E. coli onto an agarose substrate (80). Furthermore, microcontact prin ting collagen-like proteins has been shown to restrict cell growth to specified regions of surfaces (81). Microcontact printing can be used for a broad sp an of applications, however, there are still problems associated with this technique. Distorted patterns may occur due to structural deformation during contact of the surfaces and are enhanced as the dimensions of the stamp become submicron (82). These deformations are inherent due to the elastomeric nature of the polymer. Additionally, an excess of ink and the diffusion of non-covalently bound molecules to unprinted regions of the surface may cause dimensio ns of the printed pattern to become larger than those on the stamp (82). 1.3 Actin-Based Motility Actin-based motility occurs when actin filame nts elongate against the surface of a motile object, propelling the object forward to ma ke space for the addition of new monomers. Listeria a classic example of actin-based motility, forms an actin rocket tail consisting of thousands of filaments cross-linked into a bundle. Filaments in the tail elongate near the surface of the bacteria, pushing against the bacteria and propel ling it forward. Many proteins have been shown to be involved in various types of actin-based motility, including the Listeria transmembrane protein ActA and vasodilator-stimulated phosph oprotein (VASP), which ma y act together as an end-tracking motor (25, 26). Th ese and another group of protei ns (formins) have been used in vitro to replicate actin-based mo tility with bacteria and various types of particles. 1.3.1 Listeria and Particle Motility Motility of bacteria in cells, such as Listeria occurs through the recruitment of actin monomers from the host cells cytoskeleton and the subsequent generation of force through the formation and polymerization of many bundled, cro ss-linked actin filaments (actin rocket tail) at

PAGE 23

23 the rear of the bacterium (28, 29). Listeria motility has been successfully reconstituted in vitro in various cell extracts and condit ions (83, 84). Although the exact mechanism is unclear (25, 26, 85, 86), it is well-known that the surface prot ein ActA bound to the membrane of motile Listeria is a key protein required for the actin rocket ta il formation (87). Current experimental results support the role of ActA and VASP as an end-trac king motor, providing persistent attachment between the filaments and the motile object. By extracting ActA protein from Listeria polystyrene beads can be functionalized with ActA to mimic Listeria motility in vitro (88, 89). Beads provide the opportunity to study the mechanism and optimize motility by controlling important parameters, such as diameter and protein surface density of the motile object. Cameron, et al. (1999) successfully used an extraction and purification of histadine-tagged ActA from Listeria that was incubated on carboxylated polystyrene beads to coat their surf aces to replicate actin-based motility systems. They found that particle diameter affected the fraction of particles with tails and the velocity of the particles (90). Experimentally, Cameron, et al. (2001) discovered a single actin filament attached to a 50-nm bead with 37.5% surface c overage of ActA. No average velocity was reported due to thermal fluctuations of the bead and limitations of the imaging equipment (89). However, EM images showed that no 50-nm bead had more than one actin filament attached to it. The length of the actin filament attached to the 50-nm bead indicates that the (+)-end of the filament must have been attached to the bead persistently du ring elongation, otherwis e the bead would have diffused away from the actin filament during fluctuations from the surface for monomer addition (25).

PAGE 24

24 Other studies have supported end-tracking motors as the mechanism for ActA/VASP mediated actin-based motility while also demonstr ating the ability of the ActA/VASP complex to support in vitro actin-based motility of bacteria and vesicles. Kuo and McGrath were able to track the trailing ends of Listeria using an optical trapping and laser particle tracking system. They showed that Listeria moves with 5.4 nm steps with paus es in between each step. During the pauses, the bacteria fluctuated 1.31 0.0 04 nm parallel to and 0.94 0.03 nm perpendicular to the stepwise movement. This was 20-fold less fluctuation than seen by lipid droplets placed next to the bacterium in the same solution, indica ting that the actin tail must be coupled to the motile surface (91). Lipid vesicles undergoing deformation and motility due to actin polymerization was first induced by Upadhyaya, et al. (92). By incuba ting fluorescently-labeled ActA on the surface of the vesicles, ActA polarized to the end of the ve sicle where the actin tail formed. Data showing the vesicle trailing end velocity to be 2.5 m/min (six times faster than the vesicles initial velocity) at a snapping point, caused by a release of the vesicle, led to the conclusion that the actin was persistently attached to the ActA on the motile surface (92). Formins, multi-domain proteins that have been found to regulate polymerization of unbranched actin filaments, have also been used to reconstitute actin polymerization in vitro The FH1 domain of formins binds profilin (93) while the FH2 domain binds the (+)-end of the actin filament (27, 94-96). Romero, et al. adsorbed the FH1-FH2 domain from the formin mDia1 onto polystyrene beads and demonstrated a tight coupling between the polymerization and ATP hydrolysis on filament s attached to FH1-FH2 (27). In vitro both ActAand formin-induced motility provide consistent results for actin-based motility of particles, bacteria, and vesicles, all of which can be exploited in bionanodevices.

PAGE 25

25 1.3.2 Mechanisms The proteins ActA and VASP are res ponsible for actin-based motility of Listeria but currently, the mechanism as to how they produce forces is under debate. In the Brownian Ratchet model (97, 98), force is generated by actin polymerization of free-ended filaments impinging on a surface or object. The free en ergy of ATP-actin monomer addition to the filament (+)-ends is converted into the driving fo rce for the motility (99). The actin filaments are assumed not to be attached to the surface but ar e stabilized in the cyto plasm through branching and cross-linking. Thermal fluctuations of the filament (+)-e nd away from the surface allow the time and space needed for a monomer to add onto the end of the filament. Because the filament cannot diffuse backwards due to the cross-linked network, it pushes forward against the motile surface as it elongates. Based on thermodynamics, th e work done on the motile surface can not exceed the free energy released upon monomer addition, given by Equation 1-5. )crit(T T )add(][A ][A ln G kT (1-5) The Boltzmann constant and the absolute temperature, kT, represents the thermal energy, [AT](+)crit is the (+)-end critical concentration of ATP-actin, and [AT] is the total concentration of G-actin. When depolymerization at the (-)-end of the filament is occurring, the concentration of actin monomers must be less then the critical concentration at the (-)-end. Based on this concentration estimation and the length per m onomer addition of 2.7 nm, the maximum force that a filament can apply to a surface by monomer addition alone is 2 pN. This theoretical force is smaller than previously published force estimations used for steady-state elongation (86, 100). In the actoclampin model, elongating filament s are persistently attached to the surface through a multivalent surface-bound protein that in teracts with high affinity for ATP-actin

PAGE 26

26 monomers. The free energy released upon hydrolys is of ATP is assumed to drive affinity modulations between the end-tracking proteins and the filament (+)-end in a way that converts the hydrolysis energy into the mechanical work used for inserting new monomers and pushing on the motile surface. End-tracking motors may also provide some protection of elongating filament ends from capping proteins while transferring a profilin-actin complex into the filament (26). In addition to the 2 kT of energy available from th e addition of one subunit, actoclampin can utilize as much as 14 kT per subunit of free energy from ATP hydr olysis (25). Experimental results, including the dependence on profilin and ATP hydrolysis, show that formins behave like actoclampin filament endtracking motors (25-27, 101, 102). In the case of Listeria the putative actoclampin motor includes both VASP and ActA (26). VASP contains an EVH2 domain, which binds both G-actin and F-actin, and an EVH1 domain, which has binding sites on ActA. VASP also contains a proline-rich domain which is thought to recruit profilin-actin for subsequent addition onto the fi lament (+)-ends (103). Supporting this proposed role of profilin in actin-based motility by actoclampin, fluorescently-labeled profilin has been observed to localize to Listeria s surface during motility with the speed of Listeria directly proportional to the fluorescent intensity (104). 1.4 Bionanodevices Both actomyosin and a similar system using kinesin with associated microtubules have been exploited to transport material in vitro. Two different methods exist for the mechanism of transport. The first is the sliding filament assay which relies on immobilized myosin or kinesin to transport actin filaments or microtubules, respectively. The second assay is the immobilized filament assay, which binds actin filaments or microtubules to a surface to create a path for myosin or kinesin to walk along. Both have been show n to transport cargo in a directive manner.

PAGE 27

27 1.4.1 Sliding Filament Assay The sliding filament assay is more commonly used than the immobilized filament assay because it is easier to set up and execute experi ments (32). Typically, surfaces contain a region with high affinity for protein ad sorption and a region with low affi nity. Bunk, et al. found that the photoresist materials MRL-6000.1XP and ZEP-520 both efficiently adsorbed heavy meromyosin (HMM) and subsequently bound and moved actin filaments in contrast to PMMA-200, PMMA-950, and MRI-9030, which exhibi ted poor motility. For this reason, they created 100 to 200 nm grooves in a material la yer exhibiting low motility (PMMA-950) to expose an underneath layer with high motility behavior (MRL-6000.1XP) to efficiently guide actin filaments (8). Another method for surface modification includes the use of high-resolution e-beam patterning exposure of a poly[(tert-butyl-methacrylate)-co -(methyl methyl methacrylate)] to make hydrophobic (high-energy exposure) and hydrophilic (low-e nergy exposure) regions, the latter with a low affinity for HMM adsorp tion and subsequent f ilament binding (19). Microcontact printing was used to create SAMs on gold surfaces and attach biotinylated myosin to the patterned region. This method ensured the orientation of the myosin would be optimal for gliding actin filaments (18). Clemmens, et al.(2003) tested kinesin adsorption and the gui dance of microtubule motility on three different designs: a chemical border between ppPEO (low affinity for kinesin) and glass, a channel with floors and walls made from polyur ethane, and a combination of the two, using SU8-PEO photoresist to make non-adhesive walls with channel floors made of SiO2 for adsorption of kinesin. The most efficient desi gn was the latter, which combined both chemical definition and topographical boundaries (105). This design was further optimized by using AZ5214 as the non-adsorbing photoresist on glass and creating channel walls with an undercut to prevent microtubules from climbing over the sides of the channels (17).

PAGE 28

28 Sliding assays have also allo wed for the exploration of many different track shapes, which may be beneficial for sorting the direction of the moving filaments. Suzuki, et al. observed actin filaments moving in concentric ci rcles, letters, and figure eights on PMMA surfaces (21). Paths of microtubules have been studi ed in channels using different crossing junctions, including tangent circles and crosses, as well as concentric circles (16). Additionally, the sorting of microtubules has been examined by using arrow head s or tangent, straight channels shooting off of circular paths (12, 13, 16). These types of devices are designed to catch any microtubule traveling in an opposite direction in these traps and allow for the filament to turn around. The ultimate goal is for all filaments to be moving in the same direction. 1.4.2 Immobilized Filament Assay Fewer attempts have been made to immobili ze filaments for myosinor kinesin-mediated cargo transport. Glass particles attached to kinesin have successfully traveled along gluteraldehyde-immobilized, isopo lar microtubule arrays (3). Nanocrystals have also been shown to travel along microtubule arrays fixed to the surface by poly-L-lysine (5). Reuther, et al. has biotemplated microt ubules into single-molecule trac ks, which bind kinesin. These immobilized filaments successfully guided another microtubule along the path by binding kinesin to the immobilized microtubules (22). Actin paracrystal structures formed in lipid bilayers have been shown to guide myosin-coate d glass beads (6). Immobilized-filament assays have the potential to guide myosin (or kinesi n)-mediated cargo unidirec tionally by orienting the filament (+)-ends to point in the same direction (32). One attempt to align filament polarity successfully used electrostatic condensation of Factin/gelsolin to create an unidirectional track for myosin-bound bead transport (19).

PAGE 29

29 1.4.3 Material Transport An important aspect of all of these techniques is that they are able to transport material in a controlled manner. In bionanotechnology, particle s have the advantage of being functionalized to not only move by actin-based mo tility but also to bind to specifi c proteins or molecules that need to be transported against flow or diffusion gradients. As mentioned above, guidance of various particles bound to kinesin and myosin have been attempted. Quantum dots (7.6 nm diameter) functionalized with kinesin were observed m oving along microtubule tracks at an average velocity of 0.28 m/sec for distances ranging from 1 to 5 m with the number of events falling exponentially as the distance incr eased (5). Larger kinesin-coated spherical particles (1.8 to 2.8 m) made from glass, pol ystyrene, and paramagnetic polystyrene traveled along isopolar microtubules for 1 to 2.5 mm at a rate of approximately 0.5 m/sec. Suda and Ishikawa found that myosin-bound particles with 65 m diameters slide at rates of 338 m/sec on actin filament paracr ystal structures (6). Sliding actin filaments and microtubules have also been shown to carry attached cargo. Bachand, et al. showed the transport of quantum dots attached to microtubules along a kinesin-coated surface (2). Similarly, Mansson, et al. showed transport of quantum dots attached to actin filaments on a myosin-coated surface (4). This same technique has been used for motile microtubules labeled with antibody sandwiches, i.e. the microtubule can selectively bind a target antigen and then a secondary antibody for detecti on (14). Suzuki, et al. successfully transported a gelsolin-coated polystyrene bead (1 m diameter) attached to the (+)-end of an actin filament on a myosin surface (7). All of these examples demonstrate the ability to successfully transport cargo against flow and diffusion gradients th rough a variety of mechanisms, all using biomolecular motors.

PAGE 30

30 Figure 1-1. Treadmilling of an actin filament. ATP-actin binds to the (+)-end of the filament. The ATP hydrolyzes, forming an intermediate monomer in the filament, ADPPi-actin. The phosphate is eventually re leased, leaving the remaining monomers in the filament as ADP-actin. The monomers at the (-)-end dissociate from the filament and are released back into the solution, where the ADP molecule is exchanged for an ATP molecule, and the cycle repeats. Figure 1-2. Experimental set-up for binding filaments to a surface. NEM-myosin is adsorbed to a glass coverslip, where it binds a polymeri zing actin filament. A laser beam is totally internally reflected at the interf ace of the glass coverslip and the sample solution. This creates the electroma gnetic (EM) wave, known as the evanescent wave, which excites any fluorescently-labele d molecules attached to monomers that have incorporated into the actin filament.

PAGE 31

31 Figure 1-3. Schematic of microcontact printing pr otein on glass. A patterned piece of PDMS is incubated with a solution of protein. The stamp is dried and placed in contact with the glass surface. When the stamp is rem oved from the glass, the protein that was directly contacting the glass is transferred onto the glass in the corresponding pattern.

PAGE 32

32 CHAPTER 2 GUIDANCE OF ACTIN FILAMENT ELONGAT ION ON FILAMENT -BINDING TRACKS 2.1 Introduction Guidance of single actin filament elongation is a simplified system to study the potential of actin-based motility for applications in nanotechnology. By directing single actin filament elongation, controlled tracks of ac tin filaments for cargo-carrying myosin can be created. In addition, single-filament propulsion of small part icles (~50 nm in diameter) is predicted to behave in a similar way on filament-binding tracks as elongating actin filaments without particles. We developed and characterized th e ability of microcontact-printed tracks of immobilized filament-binding proteins (NEM-modified myosin) to guide the direction of filament elongation. The dire ction and rate of polymerization of individual surface-bound filaments were directly obser ved and quantified using TIRF microscopy (69, 70). Printed NEM-myosin tracks were found to capture nasc ent filaments from solution and guide their subsequent elongation to a degree that was dependent on the track width and myosin density. These findings suggest a mechanism whereby th e myosin track can recapture the tips of undulating filaments that cross track boundaries at sufficiently sma ll glancing angles. 2.2 Materials and Methods 2.2.1 Protein Preparation Actin was polymerized from a mixture of unlab eled and fluorescently-labeled G-actin. To prepare unlabeled actin, we resuspended commercial G-actin that was lyophilized as a 10 mg/mL actin solution in 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 5% sucrose and 1% dextran (Cytoskeleton, In c., Denver, CO) in 25 L deionized H2O and incubated the sample on ice for at least 2 h to dissolve the protein thor oughly. After 24-h dialysis against G-buffer (5 mM Tris-HCl pH 8.0, 0.01% NaN3, 0.1 mM CaCl2, 0.5 mM Dithiothreitol (DTT), 0.2 mM ATP),

PAGE 33

33 with three buffer exchanges, actin was collected and clarified by centrifugation at 107,000 g for 2 h at 4 C in a TLA100.2 rotor (Beckman Coulte r, Inc., Fullerton, CA). G-actin was stable at 4 C for up to two weeks. Lyophilized actin with random surface lysine residues covalently bound to an activated ester of rhodamine (Cytos keleton, Inc., Denver, CO) was used (without dialysis) at a stock solu tion concentration of 23 M after the powder was resuspended in deionized water for three days at 4 C. To prepare Alexa 488-labeled actin (73), purified rabbit muscle G-actin (106) was polymerized at a concentration of 60 M by dialysis against polymerization buffer (2 mM 3-morpholinopropanesulfonic acid (MOPS) pH 7.5, 50 mM KCl, 1 mM MgCl2, 0.2 mM ATP). A 5-fold molar excess (300 M) of Alexa Fluor 488 carboxylic acid, succinimidyl ester (Invitrogen, Molecular Probes, Carlsbad, CA) in dimethyl sulfoxide (DMSO) was added to the polymerized actin and left overn ight at 4 C. Labeled F-ac tin was then centrifuged at 288,000 g for 30 min at 4 C in the TLA100.2 rotor. G-actin was prepared from this pellet by resuspension and 48-h dialysis against G-buffer. Monomeric actin was collected and centrifuged at 107,000 g for 2 h at 4 C to remove oligomers. The monomer-rich supernatant was collected, aliquoted, rapidly frozen in liquid nitrogen, and stored in -70 C for up to six months. For actin filament binding in the absence of myosin-mediated motility, myosin II ATPase activity was inactivated by N-ethylmaleimide (NEM ) treatment, as described elsewhere (70). The contents of five tubes of commercial lyophilized 95%-pure myosin II (Cytoskeleton, Inc. Denver, CO) was resuspended in 250 L deionized H2O, yielding a 20 mg/mL myosin solution. After 2-h dialysis at 4 C against 10 mM Imid azole, pH 7.0, 10 mM ethylene diamine tetraacetic acid (EDTA), and 500 mM KCl (myosin dialysis buffer), the protein was diluted to 10 M in the same buffer, and reacted with 1 mM of NEM for 1 h on ice. DTT (1 mM) was used to quench

PAGE 34

34 the unreacted NEM for an additional 1 h on ice. After overnight dialysis at 4 C against 10 mM Imidazole, pH 7.0, 500 mM KCl, 10 mM EDTA and 1 mM DTT, with one additional buffer exchange, NEM-myosin was aliquoted, rapidly frozen in liquid nitrogen, and stored at -70 C for up to six months. Purified, precipitated profilin expressed from Escherichia coli and stored in lysis buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT, and 1 mM phenylmethanesulphonyl-fluoride (PMSF)) in the presence of 40 mM NaCl was generously donated by Dr. William Zeile. Profili n precipitate was spun at 16,000 g for 20 min in an Eppendorf 5415C microcentrifuge, resuspended in 4 polymerization buffer (20 mM Imidazole pH 7.0, 200 mM KCl, 4 mM MgCl2, and 4 mM ethylene glycol te traacetic acid (EGTA)), and dialyzed for 48 h against the same buffer at 4 C. 2.2.2 Microcontact Printing NEM-myosin was microcontact-printed as para llel tracks on glass coverslips with the aid of a polydimethylsiloxane (PDMS) stamp. A s ilicon wafer mold of desired pattern was created by light exposure of SU8 photores ist (Microchem Corporation, Ne wton, MA) using a Karl Sss MA-6 photoresist microlithographer. A mixture of 10:1 prepolymer to catalyst of Sylgard 184 (Dow Corning Corporation, Midland, MI) was de gassed and poured onto the surface-fluorinated silicon wafer, after which PDMS was polymerized in the mold at 60 C for 1 h (107). The PDMS stamps were generously made and donated by Dr. Suzanne Ciftan-Hens (International Technology Center, Raleigh, NC). The resulting hydrophobic PDMS stamps (with tracks ranging from 3 20 m wide) were inked for 40 min with a NEM-myosin solution, wash ed three times with myosin dialysis buffer, three times with deionized H2O, and dried under nitrogen (79, 107). After acid-washing and treatment as described elsewhere (70), glass cove rslips (No. 1, Fisher Scientific, Waltham, MA)

PAGE 35

35 were pressed and left in contact with a stamp s inked-surface for 20 min. The resulting stamped coverslips were either examined by atomic force microscopy (AFM Dimension 3100, Veeco, Woodbury, NY) or used in hand-fabricated flow -cells constructed by adhering the inverted coverslip onto a microscope slide with double-si ded Scotch tape. Until use, flow-cells were stored in myosin dialysis buffer for up to 6 h. 2.2.3 Actin Polymerization Actin filaments were polymerized according to a previously publis hed protocol (70). Actin monomer solution was combined with a 10 magnesium-exchange buffer (10 mM EDTA, 1 mM MgCl2) and incubated on ice for 5 min to convert Ca-ATP-actin to Mg-ATP-actin. The Mg-ATP-actin was then mixed with a 2 concen trated polymerization buffer (20 mM Imidazole pH 7.0, 100 mM KCl, 2 mM MgCl2, 2 mM Ethyleneglycol-bis( -aminoethyl)N,N,N ,N -tetraacetic Acid (EGTA), 20 mM DTT, 0.4 mM ATP, 1% methylcellulose, 30 mM glucose, 40 g/mL catalase and 200 g/mL glucose oxidase) for a final concentration of 0.75 to 1.5 M actin (15-30% fluorescently-labeled). This solution was immediately transferred into the flow-cell. Prior to experiments, flow-ce lls containing cleaned glass cove rslips were treated according to Kuhn and Pollard (70), with 0.2 M NEM-myosin for 1 min followed by 1% bovine serum albumin (BSA) in 50 mM Tris-HCl, pH 7.6, 600 mM NaCl (HS-TBS) to remove any unbound NEM-myosin, followed by washing with 1% BSA in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl (LS-TBS) to remove any unbound NEM-myosin, lo wer the salt concentr ation in the flow chamber, and to passivate any exposed glass ( 70). The volume of each wash was approximately twice the chamber volume. Flowcells possessing NEM-myosin-stamped coverslips were washed with the 1% BSA in 50 mM Tris-HCl, pH 7.6, 600 mM NaCl and 1% BS A in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl before actin solution was added.

PAGE 36

36 2.2.4 TIRF Microscopy and Data Analysis To observe our samples, we used a commer cial objective-based TIRF consisting of a Nikon Eclipse TE1200 inverted microscope fitt ed with TIRF optics (Nikon Instruments, Melville, NY) and an Argon 488-nm or a Helium-Neon 532-nm laser (Melles Griot, Carlsbad, CA). Images were acquired using a digital charge-coupled device (CCD) camera (QImaging, Burnaby, BC, Canada) and analyzed using MetaMorph software (Molecular Devices Corporation, Sunnyvale, CA). Time-lapse images of each field-of-view were taken at 5-sec intervals. To quantify filament orientation, the filament images were first discretized into segments by selecting points spaced 0.1 3 m apart along the filament lengt h. We only analyzed those filaments that were elongating during the obs ervation time. Filaments that spanned the NEM-myosin track border were discretized up un til the track edge. Furthermore, to eliminate the possibility that interacting filaments might bi as the data, images containing a high density of elongating and non-elongating filame nts near the surface were excluded. The minimum number of independent tracks for each track width was ten, while each track width contained measurements from at least 84 filaments. The minimum number of independent tracks for one NEM-myosin treatment concentration was ten, while each condition contained measurements from at least 86 filaments. Taking as the angle between the segment and th e track direction, the mean of cos(2 ) (weighted by segment lengths) for each fila ment was calculated. A mean of cos(2 ) equal to one corresponds to perfect alignment, and a value of zero indicates random alignment. An overall cos(2 ) for each track width was cal culated by averaging the cos(2 ) values of the filaments, weighted by the filament length. Data were co llected from samples run on different days, but a one-way ANOVA between the samples for each tr ack width concluded that no statistical

PAGE 37

37 differences were present between days. Theref ore, each individual track was considered an independent sample and was used to determin e the standard error for each condition. The weighted mean cos(2 ) was plotted against both track widths and NEM-myosin treatment concentrations with error bars repr esenting the standard error (SE) based on a weighted standard deviation (SD). To dete rmine if the alignment of the filaments was significant, a one-sample t-test was used to calculate a one-tailed p-value by comparing the average cos(2 ) for each track width and NEM-myos in concentration with a cos(2 ) value of zero for random alignment. The highest and lowe st NEM-myosin treatment concentrations were compared using a two-sample t-test. Filaments within 1.5 m of the track edge were pooled to obtain alignment near the edge of the track. In addition, the average di stance of each filament segment from the edge of the track was estim ated based on positions obtained manually from MetaMorph, and the distances were plotted agai nst the alignment for each segment to create scatter plots for all samples. Changes in filament length were measur ed for a minimum of three time-points per filament, allowing us to calculate the elongati on rate of filaments for each NEM-myosin and profilin concentration from a line-fit of filament length versus time (with each 0-time intercept set to 0). Instantaneous elongation rates, with subunits/sec values (based on 370 subunits per m filament length), were pooled from at least 12 filaments for each NEM-myosin concentration and at least 13 filaments for each profilin concentr ation. Elongation was observed and measured at filament (+)-ends only. Elongation rates for th e four NEM-myosin treatment concentrations were tested for statistical diffe rences using a one-way ANOVA.

PAGE 38

38 2.3 Results 2.3.1 Filament Binding to Stamped Surfaces Using the microcontact-printing procedure, we produced and characterized filament growth on myosin tracks of six different average wi dths SD (3.3 0.3, 4.3 0.5, 5.7 0.2, 10.7 1, 15.1 0.8, 20.2 0.5 m). Fluorescence and AFM images were taken to assess the uniformity of NEM-myosin coverage. As shown in Figure 2-1A, the NEM-myosin regions were clearly delineated by the preferential non-specific adsorption of fluorescent actin monomers or oligomers with the NEM-myosin regions re lative to the BSA regions. The magnified three-dimensional AFM image (Figure 2-1B) of a representative track edge shows a confluent protein layer, with thickness shown in Figure 21C. The measured thickness variation of the protein layer is comparable to the 7-nm diamet er of a single actin filament, implying that the peaks and valleys of the layer should have little effect on the guidance of the filaments. The microcontact-printed surfaces were exposed to the actin solution, allowing capture of nascent filaments from solution by the NEM-myosin-coated regions with the majority of captured filaments continuing to elongate on th e surface. (Surfaces stamped with 0.5 mg/mL BSA showed little evidence of filament binding, as shown in Fi gure 2-2.) We were able to monitor (+)-end filament elonga tion on the NEM-myosin tracks in real-time using TIRF microscopy (Figure 2-3). Notably, the tips of myosin-bound filaments clearly undulate during elongation, and the filaments continue to bind to NEM-myosin along their length as they extend. While filament segments were found both on the tr acks and on the BSA-coated regions, filament segments on the BSA regions underwent larger thermal undulations (observed in the xand y-direction) than those confined on the NEM-myosin track, indicating that filaments were tightly bound only to the NEM-myosin tracks (Figure 2-4). Filaments initially bound to the track either remained in the NEM-myosin region or elongate d beyond the track edge, with the filament end

PAGE 39

39 continuing to elongate and undulate over the BSA-coated region (Figure 2-5). In many cases, these undulations allowed a filament which had crossed the boundary of the NEM-myosin track at a glancing angle to rebind to the track, now with the filament end often aligned with the track edge (Figure 2-6). 2.3.2 Alignment of Filaments To determine the effect of the NEM-myosin tracks on the filament alignment, we first eliminated samples containing a high density of actin filaments on the surface in the track region (Figure 2-7). The high density of filaments near the surface may increase filament-filament interactions, which could then increase the alignm ent of the filaments with each other. Also, the dense samples were difficult to analyze becau se individual filaments were not always distinguishable. The resulting alignment of the filaments with the track edge was quantified by measuring the angle for segments along each of several filaments for each track width (Figure 2-8A) and calculating the mean of cos(2 ). Filament alignment on the narrower track widths was found to be significant and the degree of alignment increa sed as the track width decreased (Figure 2-8B, solid squares). To determine whether alignment resulted primarily from interactions with track boundaries, the mean of cos(2 ) was re-calculated for the subset of filaments within approximately 1.5 m of the track edge on tracks wider than 3.3 m (Figure 2-8B, open squares). The filaments near the track boundaries had a similar degree of alignment independent of the track width. These observed trends are cons istent with the interpretation that filaments in tracks with widths comparable to th e filament persistence length (~ 10 m (48, 49)) take random walk trajectories within the boundaries of the track and only become aligned when encountering the track boundary. On the other hand, filaments in tracks that are small re lative to the filament

PAGE 40

40 persistence length encounter the track boundary and become realigned before significant changes in direction occur (Fig ure 2-8C). The probability of realignment of a filament with the track edge is also dependent on the alignment of the filament immediately before crossing the NEM-myosin track edge. The histogram in Figure 2-9 demonstrates that at smaller alignment angles, these rebinding events were more frequent, ranging from ~90% reca pture-probability for angles less than 15 decreasing to zero probability at a ngles greater than 60. From this result, we surmise that the bending energy at large angles required to recaptu re a filament is too large to be achieved by thermal undulations. To confirm that thermal undul ations caused this bending, we estimated the bending energy of several filaments from Equation 2-1 (108). ds ds d 2 TkE2 B bend (2-1) The thermal energy is kBT (Boltzmann constant multiplied by temperature), is the filament persistence length (~ 10 m (48, 49)), and s is the arc length of a filament that crossed the boundary and then bent back to be ca ptured by the track. In each case, Ebend was on the order of kBT suggesting that changes in filament dir ection were driven by thermal energy (49). A scatter plot shows all of the filament segments and their alignment as a function of distance from the edge of the track (Figure 2-10). Segments seem to be concentrated near the edge at both aligned (cos(2 ) = 1) and unaligned angles (cos(2 ) = -1). This suggests when filaments reach the edge of the track, they tend to realign at small angles, increasing alignment, or they leave the track completely at large angl es, making the distribution of segment angles near the track edges at the extremities.

PAGE 41

41 2.3.3 NEM-Myosin Concentration We also investigated the effect of NEM-m yosin concentration used to treat the PDMS stamps on the alignment of elongating filaments. As shown in Figure 2-11A, the degree of alignment, again quantified by the mean cos(2 ), trended upward with NEM-myosin treatment concentration, with the degree of alignment on tr acks prepared with the highest concentration tested (2 M) significantly greater than that of the lowest (0.1 M), the latter of which failed to generate statistically significant alignment. These results suggest that lower NEM-myosin surface densities are less efficient at recapturing undulating filament ends that have elongated off the track edge. Finally, we anticipated that at higher densities, myosin might bind at or near the filament (+)-ends and thereby hinder monomer incorporation. To determine the effect of myosin concentration, we measured the elongation rate of filaments on the protein tracks prepared with varying NEM-myosin concentrations used in Fi gure 2-11A. As shown in Figure 2-11B, the elongation rate decreased only slightly for the hi ghest myosin concentration, implying that the NEM-myosin had little effect on actin polymerization. Scatter plots of segment distance from the tr ack edge versus filament alignment for each NEM-myosin concentration are shown in Figure 2-12. The 0.1 M NEM-myosin treated sample has the least bias of filament se gments aligned near th e edge of the track, presumably due to the low concentration of myosin available to re bind and realign filaments. The 0.5 and 2 M samples show a slight bias toward aligned filament segments. The 1 M sample, which had the most segments available for analysis, is the subset of the filaments in Figure 2-10 that were on a 5 m wide track. Therefore, the 1 M sample shows the same trend as Figure 2-10, again indicating that the alignment of filament segments closer to the track edges is independent of track width.

PAGE 42

42 2.3.4 Control of Actin Polymerization Figure 2-13 shows a sample in which filaments accumulated within the BSA regions (spaces where NEM-myosin was not stamped). Although these filaments undergo large thermal undulations, unlike those that are bound onto the NEM-myosin tracks, their accumulation seems to prevent filaments from binding to the NEM-myosin track. These interactions could be due to NEM-myosin molecules that adsorbed onto an area that was not within the stamp, interactions between actin filaments, nonspecifi c interactions between actin filaments and BSA or glass, or a combination of all three effects. The density of filaments in these regions is observed to increase over time, presumably because of the strong effe cts actin filaments have on each other. In addition, if the filaments are diffusing to the su rface as long filaments (lengths greater than the track width), the filaments at most may lay across the track at some angle rather than align with the track. One way to potentially prevent this behavior is to control actin polymerization through nucleation to prevent filaments from accumulating near the surface within a short time frame. Protein samples may contain small oligomers that cause the density of filaments to increase at much higher rates than other samples. Also, the lyophilized rhodamine actin used contains groups of actin monomers that have not completely dissolved, causing them to behave as potential nuclei. Figure 2-14 shows how the actin filament density is de creased by increasing the resuspension time of the lyophilized rhodamine actin, allowing for the breakdown of the aggregates of actin monomers. In an attempt to control the polymerization further, profilin was mixed in with the polymerization assay at con centrations between 1 and 20 M. On surfaces treated with NEM-myosin, profilin clearly had an effect on the filament density near the surface (Figure 2-15). The elongation rate of filaments exposed to varying levels of profilin decreased only

PAGE 43

43 slightly as the profilin concentration increased (Fi gure 2-16). This indicates that the decrease in filament density near the surface is mostly due to the sequestering of monomers of actin, thus preventing nuclei from forming. The decrease in elongation rate may have only a slight effect on the density. Although the decrease in density was apparent on NEM-myosin surfaces, when attempting to use profilin in an assay with stamped surfaces, the amount of labeled actin monomers that did not incorporate into filaments created a high background on the surface, making visualization of elongating filame nts over time difficult to achieve. 2.4 Discussion The data presented demonstrates that microcont act printing can be used as an effective way for guiding the polymerization of actin filament s. By printing NEM-myosin tracks on glass surfaces, actin filament binding was confined to certain regions of a substratum and aligned by controlling the path of filament elongation. Smalle r track widths provided the greatest degree of alignment by increasing the frequency of elonga ting ends encountering the track boundaries, whereupon the filament elongation di rection tended to preferentially align with the edge of the NEM-myosin tracks, and alignment appeared to be confined primarily to the track boundaries. The degree of alignment, but not the polymeri zation rate, depended on the concentration of NEM-myosin used to prepare the stamp, with the lowest concentration (0.1 M) generating little, if any, alignment. Notably, this lower value is near the minimum concentration needed to provide a monolayer on the PDMS stamp (20), hence it is likely that, in this case, less than a confluent layer was transferred upon printing. 2.4.1 Mechanism for Filament Alignment Taken together, our results can be explained by the following alignment mechanism (Figure 2-17). Nascent filaments are captu red on the track surface and elongate on the NEM-myosin. As a filament elongates, the ne wly polymerized portion is quickly captured by

PAGE 44

44 the myosin surface until the filament end reaches the NEM-myosin track boundary. The filament continues to grow across the boundary wh ile unattached to the substratum and becomes capable of undergoing larger th ermal undulations. These undulations allow the filament to be recaptured to the track in a more aligned direct ion, provided the angle at which the filament encounters the track boundary is sufficiently small, and the myosin surface density is sufficiently large to allow NEM-myosin in the track to rebind the undulating filament end. Clemmens, et al. previously demonstrated a similar tendency of microtubules gliding along a kinesin-coated surface to align at a chemically-defined border (105). They found that microtubules began to undulate as they crossed a border from kinesin-ri ch to kinesin-poor surface regions and that their fluctuations were sufficient to re-align the gliding microtubules at small approach angles. Furthermore, Sundberg et al. observed a comparable trend when analyzing the guidance of gliding actin filaments with respect to their approach angle on myosin tracks with a chemically-defined border (15). While similar, the mechanism in our study differs in that the filament lengths are bound and do not slide relative the substratum, and the elongating filament end moves by undulating about a fixed mean position determined by orientation and position of the immobilized portion of the filament. Our findings suggest that at narrower track widths than measured here, even greater alignment would be achieved, with greatest alig nment occurring when the myosin track widths approach the filament width, similar to what wa s previously observed with sliding microtubules on kinesin tracks (provided that the track of ki nesin was no wider than the microtubule itself) (22). Also similar to our observed effect of track edges on elongation direc tion, Nicolau, et al. observed an increased alignment of (non-elongati ng) actin filaments gliding by ATP-dependent myosin translocation near the boundary of regions with different myosin density (19).

PAGE 45

45 2.4.2 Potential and Applications Our results indicate that filament-binding tracks are a viable method for guiding the direction of filament elongation on surfaces in vitro A potential application is laying single-filament tracks for myosin-based transpor t of nanoor microparticles (i.e., molecular shuttles) (10). One advantage of this techni que over other methods fo r depositing filaments on patterns (e.g. electrophoresis and flow) is that the polymerization of i ndividual filaments could be potentially guided to follow tracks with comp lex patterns (e.g., with curves and bends, without sharp angles), thereby achieving more complex systems and networks of myosin-mediated transport. Moreover, after th e initial segment is deposited (perhaps with position and orientation cont rolled by flow or electrophoresis ( 23, 32, 109)), the polarity of the generated filament track would be controlled by its (+)-end-only elongati on, thereby ensuring the proper orientation for unidirectionally -guided myosin-based shuttles. F-actin binding tracks can poten tially guide elongation of individual long filaments or multiple interconnected shorter filaments ove r long distances (hundreds of microns) for long-distance transport of nanoand micro-scal e cargo. The filament density depends on the filament nucleation rate and can be controlled bi ochemically (e.g., by adding profilin). We have shown that profilin reduces the density of actin fi laments near the surface w ith little effect on the elongation rate. Optimization of the microcont act printing procedures and actin filament polymerization assay can provide control over not only filament density, but also filament elongation rates, total length per filament, and n on-specific interactions between filaments and the low-affinity surfaces (BSA and glass). A second class of potential applications for oriented filament elongation could be the guidance of molecular shuttles boun d to the elongating filament (+ )-ends, rather than relying on myosin-based transport of gliding filaments boun d to cargo (4, 7). End-tracking proteins are

PAGE 46

46 capable of linking the elongating actin filament e nd to a particle and facilitating insertional polymerization of the tethered end (25-27) Because elongation is guided by filament undulations and filament binding to the track, we would expect similar guidance of elongating filaments with or without partic les bound to the filament ends, provided the particles are small enough not to interfere with the undulating end. In summary, guided elongation of free filament (+)-ends represents an additional tool for transporting molecular shuttles, either by laying tracks in specific patterns for myosin-based shuttles, or by guiding propulsion of particles bound to filament (+ )-ends. Further investigation of surface properties, including other methods of binding filaments, density of filament-binding molecules, and varying track shapes, may provi de additional control over the alignment and guidance of polymerizing actin filaments. This approach, which relies on filament elongation rather than filament transport, should comple ment other published methods for actin alignment and actin-based molecular shuttles (6, 23-24) and broaden the methods available for transport in bio-based nano-devices.

PAGE 47

47 Figure 2-1. Images of microcontact-printed myosin tracks on a glass coverslip. A) TIRF image of NEM-myosin track (brighter region slightly right of center) delineated by nonspecific adsorption of fluorescently-labeled actin monomers to adsorbed NEM-myosin (scale bar = 10 m). B) Topographic AFM image of track edge showing uniformity of NEM-myosin coverage. C) Line-scan of vertical displacement versus horizontal displacement from plot B. The height of the step-edge was found to be approximately 8 nm and the peak-to-valle y height variations in the NEM-myosin region were found to be on the order of 4.4 nm.

PAGE 48

48 Figure 2-2. Time-lapse image of actin fila ments on a BSA-stamped surface containing no pattern. Filaments did not bind to the surface, and many fluctuated into and out of the 200 nm TIRF region. The first frame is 20 seconds after the time-lapse began, which was approximately 1 to 2 minutes after po lymerization was initiated (0.75 M actin, 30% rhodamine-labeled actin, scale bar = 10 m).

PAGE 49

49 Figure 2-3. Total internal reflection fluorescence microscopy images of elongating actin filaments on NEM-myosin tracks of approximately 3.7 m (A) and 19.5 m (B) widths. Images were taken approximate ly 48 min and 22 min after initiation of polymerization, respectively (1.5 M actin, 15% Alexa 488-labe led actin, scale bars = 5 m). Figure 2-4. Filaments in BSA region undergo la rge thermal undulations compared to filaments bound to NEM-myosin region. The NEM-myosin region, located in the top half of the field-of-view as indicated by the bri ghter background, binds filaments to the surface as they elongate, while filaments in the BSA-passivated region (bottom half) continue to leave and enter the field-of-vie w, indicating that they are not bound to the surface (scale bar = 5 m).

PAGE 50

50 Figure 2-5. Total internal reflection fluorescence microscopy im ages of undulating ends of elongating filaments. A) Images from a time-lapse sequence for a filament end elongating within a NEM-myosin track (time interval = 195 sec; da shed-line indicates track edge). B) Composite of images from A, indicating small undulations away from the ultimate filament trajectory when elongating on NEM-myosin. C) Images from a time-lapse sequence for a filament elongating across the track edge and into the BSA region (time interval = 100 sec). D) Composite of images from C, indicating larger undulations of the filament end ove r the non-binding BSA surface (scale bars = 2 m). Figure 2-6. Filament elongating passed the track edge at small A filament binds initially to the NEM-myosin track (top half of field-of-view) and begins to undulate, binding down as it elongates, until it eventually a pproaches the track edge. The filament leaves the track edge at small and undulates over the BSA-passivated region (bottom half). The undulations are even tually large enough that the filament encounters the NEM-myosin track and rebinds in a way that aligns the filament with the track edge (scale bar = 2 m).

PAGE 51

51 Figure 2-7. Filament alignment as a function of filament density on tracks. Each data point on the graph represents the average alignm ent of each field-of-view for the 20 m wide tracks as a function of the density of all filaments within the track boundaries. Any density higher than 0.4 m filament length/ m2 was eliminated due to the potential of filament-filament interactions interfering with the alignment of the filaments on the tracks. B) A field-of-view with a low density of filaments allows observation of interactions between filament s and the modified surfaces only. C) A field-of-view with a high density of filaments (density > 0.5) inhibits analysis of the sample and may include other interactions. The tracks in the high density sample are between the first two lines and the second two lines (scale bars = 10 m).

PAGE 52

52 Figure 2-8. Effect of track wi dth on filament alignment. A) Example of filament segmentation to estimate orientation angle with track edge. Left: Original TIRFM image; Middle: Same image showing filament segments ; Right: Enlargement of boxed region in middle image showing how was determined for each individual segment. Scale bar = 2 m. B) Solid squares represent the degree of filament alignment with NEM-myosin tracks versus track width. The error bars represent the weighted standard error among independent samples fo r each track width. Significant filament alignment was present in the three narrowest track widths (3.3 m: n = 16, p = 0.005; 4.3 m: n = 36, p < 0.0001; 5.7 m: n = 14, p = 0.042; 10.7 m: n = 17, p = 0.11; 15.1 m: n = 19, p = 0.14; 20.2 m: n = 10, p = 0.051). Open squares represent the alignment of filaments within approximately 1.5 m of the track edge (4.3 m: n = 36, p < 0.0001; 5.7 m: n = 14, p = 0.006; 10.7 m: n = 14, p = 0.001; 15.1 m: n = 13, p = 0.036; 20.2 m: n = 7, p = 0.038). C) Time-lapse sequence showing the alignment process for an elongating filament (+)-end at the track edge (scale bar = 2 m).

PAGE 53

53 Figure 2-8. Continued

PAGE 54

54 Figure 2-9. Histogram showing the fraction, f of filament ends that rebind to tracks after their elongating ends cross track boundaries with va rying initial angle of incidence with track edge. Error bars repr esent standard error = ( f ( 1 f )/N )1/2, for N total filaments observed within each range of angles (105). Note: No filaments crossing the track at > 60 were observed to rebind to the NEM-myosin track.

PAGE 55

55 Figure 2-10. Scatter plot showi ng each segment alignment with th e track edge as a function of the distance from the track edge. The distance of each segment was estimated by taking the midpoint of the segment and calcu lating its distance from the track edge. The distribution of alignment is bimodal at the edge, indicating that the filaments tend to align at the track edge if their alignment angle is sufficiently small. Note that the highest concentration of segm ents occurs between 0 and 3 m of the track edge (n = 4374 segments for all track widths at 1 M NEM-myosin concentration).

PAGE 56

56 Figure 2-11. Dependence of filament alignmen t and elongation rate on the concentration of NEM-myosin used for microcontact prin ting (average track width = 4.6 0.6 m). A) Alignment appears to decrease for 0.1 M NEM-myosin treatment condition. The error bars represent the standard error among independent samples for each myosin concentration. All treatments except 0.1 M NEM-myosin provided significant alignment of filaments (n = number of tracks, p = one-tailed p-value). Additionally, the 2 M and 0.1 M samples were statistically differe nt from each other (p = 0.021). B) Filament elongation rate is insensitive to changes in NEM-myosin concentration used in printing (0.75 M actin, 15% rhodamine-labeled actin; n = number of filaments that instantaneous velocities we re measured, p-value = 0.17 from a one-way ANOVA). The error bars represent the standa rd error from a least squares regression analysis.

PAGE 57

57 Figure 2-12. Scatter plots showi ng the alignment of individual f ilament segments (independent of length) at varying NEM-myosin concentr ations as a function of the distance from the track edge. A) The 0.1 M NEM-myosin treatment had the least number of segments, and the alignment does not seem to have a strong dependence on the edge of the track (n = 365 segments). B) NEM-myosin concentration of 0.5 M has a slightly higher density of aligned filaments (n = 524). C) 1 M NEM-myosin has a similar trend to Figure 2-10 (n = 1583). D) A majority of segments near the edge are highly aligned in the 2 M NEM-myosin concentration sample (n = 487).

PAGE 58

58 Figure 2-12. Continued

PAGE 59

59 Figure 2-13. Filaments accumulate in the BSA region of the stamped surface, preventing binding from occurring in the track region. Surface stamped with 2 M NEM-myosin treated PDMS stamp. Image taken 11.5 min after initiation of polymerization (scale bar = 10 m). Figure 2-14. Incubation time of l yophilized rhodamine actin can he lp to control the density of filaments on the surface. Surfaces have b een incubated with NEM-myosin for 1 min (no stamping) followed by BSA. A) Lyophilized rhodamine actin resuspended and incubated for 3 hours produces a large netw ork of filaments within 2.5 min of polymerization. B) Resuspension and incuba tion of lyophilized rhodamine for 3 days before used in a polymerization assay visi bly decreases the filament density on an NEM-myosin surface (time = 6.5 min). Both samples contain 1 M actin with 15% labeled (scale bars = 10 m).

PAGE 60

60 Figure 2-15. Increasing the concentration of profilin visibly decreases the density of actin filaments on NEM-myosin treated surfaces. A) 0 M, B) 1 M, C) 5 M, D) 10 M, and E) 20 M profilin added to the polymerization assay before initiation of polymerization. Images were taken approximately 15 minutes after initiation of polymerization (scale bar = 5 m).

PAGE 61

61 Figure 2-16. Elongation rate of actin filament s as a function of profilin concentration. Elongation rate of filaments decreases slightly with increasing profilin concentration. Error bars represent standard error based on a least squares analysis, and n refers to the number of filaments for each concentr ation observed. Elongation rates were obtained for each condition from a best-fit line of change in length versus change in time.

PAGE 62

62 Figure 2-17. Illustration of the likely mechanism for actin filament alignment on NEM-myosin tracks. Elongating filament ends first encount er and then undulate over the track edge. The increase in filament length over the track causes larger undul ations to occur. Finally, the filament either is recaptured by the track for smaller (A), or continues to elongate away from the track for larger (B).

PAGE 63

63 CHAPTER 3 SIMULATING ACTIN FILAMENT ELON GAT ION ON MODIFIED SURFACES 3.1 Introduction We have developed a simulation that allows us to predict the effect specific actin and surface parameters (filament length, filament persistence length, myosin surface binding probability, and patterned surface track width) have on filament alignment. These simulations complement our experimental results and aid in designing patterns to guide actin filament elongation in applications. Mathematical modeli ng of thermal undulations of actin filaments in solution (with both ends free) ha s been previously reported (49) We simulated the thermal excitations of the bending modes of the undulating unbound elongating filament end (with a fixed (-)-end) to generate realiza tions of the filament shape (49, 108). These simulations allowed us to examine the effects of total filament le ngth, binding probability, and persistence length on the probability of filaments rebinding after crossing a track boundary. We further investigated the effect of NEM-myosin track width, binding probability, and persistence length on the overall alignment of filaments. 3.2 Methodology The bending energy, E of a filament undulating around a straight shape is used to determine the fluctuations of the filament by calculating the shape of the filament at an instantaneous point (Equ ation 3-1) (108). 2 L 0ds(s) 2 kT E (3-1) Bending energy is a function of the pers istence length of an actin filament, the length of the filament segment, L, the Boltzmann constant, k, the temperature, T and the tangent angle, at position s on the filament. The tangent angle can be written as a Fourier series (49), with each

PAGE 64

64 mode obeying the boundary conditions: (0)=0 (fixed end) and (L)=0 (free end) (Equation 3-2). L s 2 1 n N 0n) (nsina (s) (3-2) where N is the number of modes, an is the amplitude of the wave function for modes n. The integral of ( (s))2 from s = 0 to s = L was solved for from Equation 3-2 and substituted in Equation 3-1 resulting in Equation 3-3. 0n n 0n 2 1 2 nE)(na 4L kT E (3-3) According to the equipartition theorem, the average energy for each mode is equal to kT/2, allowing us to set the energy equal to this value (Equation 3-4). 2 kT a)(n 4L kT E2 n 2 1 n (3-4) Rearranging Equation 3-4, we solved for the mean square amplitudes as a function of n, L and (Equation 3-5). ) (n 2L a2 1 2 n (3-5) Undulations of the free end of a filament were simulated by providing random values for the amplitudes an from a Gaussian distribution (MA TLAB function RANDN) with mean zero and variance given by Equation 3-5. The instantaneous filame nt tangent angles, (s), were then determined by substituting the generated amplitudes into Equation 3-2. The realized function (s) was converted into xand y-coordi nates by Equations 3-6 and 3-7. s cos s x (3-6)

PAGE 65

65 s sin s y (3-7) The coordinates x(s) and y(s) were finally obtain ed by numerical integration (trapezoid rule) of Equations 3-6 and 3-7. Elongation and binding of the filament on a filament-binding surface was simulated as follows. The free end of the filament, with the in itial length taken as 0, increased in length by s over each time increment, t (unless otherwise specified, s =0.01 m and t = 1 s). The probability of attachment along the unbound undulat ing segment length was assumed uniform along the filament length and constant in time such that the probability of binding was KpL t, where the parameter Kp is the binding probability per unit length, per unit time (m-1sec-1). Therefore, within the simulation, binding of a filament with le ngth L occurred in time increment t when MATLAB function RAND, which generate s uniformly distributed random numbers on [0,1], generated a random number < KpL t. If the filament binds, the position of binding s1 is then determined from a uniform distributi on (again using MATLAB function RAND) along the length of the free end [0, L], and the resulti ng shape of the newly bound filament segment is assumed to be fixed and is taken as the inst antaneous filament shape for that region [0 s1] (based on Equations 3-6 and 3-7). After binding, the b ound position on the filament resets as s = 0 and L is reset to the new length of the unbound filament end. Filaments were simulated on patterned surf aces with binding and non-binding regions by allowing binding to occur only on the filament-binding regions of the surface. The filament was assigned an initial position within the track, an initial angle between 0 and 90, and a final total length between 1 and 16 m, all based on a random number genera tor (MATLAB function RAND, unless otherwise specified ). In addition, the track width of the NEM-myosin, the

PAGE 66

66 number of modes, th e binding probability, Kp (m-1sec-1), and the persistence length, (m) was set by the user. The Appendix contai ns the MATLAB code with comments. 3.3 Results 3.3.1 Description of Simulated Filament Elongation To first confirm that the simulations agree qua litatively with our expe rimental observations of undulating filament ends near track boundaries we looked at the a lignment of elongating filaments restricted to the track edge (see Figure 2-9 for the experimental results). Filaments were initially bound at their (-)-end 0.01 m from the edge of the track, a nd the initial angle was determined to be between 0 and 90 by a random number generator in MATLAB (function RAND). The initial position of 0.01 m from the track edge was used to ensure that filaments crossed the track edge at some point during their elonga tion, even at small angles. The initial angle of the filament was assumed to be the an gle of incidence, or the angle at which the filament crossed the track boundary. Filaments were then set to elongate at a rate of 0.01 m/sec. The position of the filament (+)-end with resp ect to time shows the fluctuations of the filament as it was elongating (Figure 3-1). Figure 3-1A clearly s hows the decrease in fluctuations of an elongating fila ment end due to the decrease in the length of the free filament. The position at which the decrease in fluctuat ions occurred corresponds to the position on the final filament where binding occu rred (Figure 3-1B). Figure 3-1C shows the fluctuations of a filament that never rebinds to the track. These fluctuations continued to increase in magnitude. Figure 3-1D shows the unbound filaments instantane ous position at the end of the simulation. We analyzed the persistence length of filament s using Equation 3-8 (48) to confirm that the measured value of th e persistence length, was comparable to the input value.

PAGE 67

67 2 s exps ss cos)C(sj jji i (3-8) To do so, we pooled data from 240 simulated f ilaments and estimated th e angle between points at distances 0.01 m apart on the filament. B ecause we had a large sample of filaments to analyze, we only used the case where sj = si+1. We then plotted the average jjis ss cosln versus sj (for sj = 0.01 to 2 m) and found th e slope of the best-fit line with an intercept set to 0. The calculated persistence length of approximately 12 m (R2 = 0.996), matched reasonably we ll to our input value of 10 m. At a lower resolution (0.1 m segments) with the same conditions, the persistence length for 1750 filaments was approximately 14 m (R2 = 0.998). Both of these values correspond to filament shapes calculated with 2 modes. When the number of m odes was increased to 10, the persistence length was calculated (at the lower resolution) to be ap proximately 13.5 m, showi ng little effect of the number of modes on the persistence length of the filament. 3.3.2 Probability of Filament Rebinding From our experiments (cf. Chapter 2), we e xpected filaments with smaller angles of incidence to have a higher pr obability of rebinding to th e track edge (Figure 2-9). Experimentally, many characteristics of the filame nts were not constant, including the lengths of filaments, the angles that the filaments crossed the track edge, and the pos ition of the filaments with respect to the position of the track edge. Fo r our first set of simulations, we kept the initial filament position the same for every filament and then calculated the probab ility of the filament rebinding once it crossed the track edge as a func tion of the angle of inci dence. Error bars on graphs showing the probability of rebinding were calculated as de scribed in Figure 2-9 ( standard error = (f(1-f)/N)1/2)).

PAGE 68

68 Figure 3-2 shows the effect of decreasing the time, t, of the simulation from 1 to 0.5 seconds with a constant elong ation rate on the proba bility of the rebinding of 3.2 m long filaments (average length of filaments for our experiments). As expected, there was little variation in the dependence of the rebinding prob ability on the incidence angle. The largest difference occurs between 20 and 30, which is most likely a region of higher variability, based on the larger error bars. We next looked at the effect of the rebinding probability as a function of the number of modes used to simulate the filament shape. Figure 3-3 shows very little variation and no apparent trend between th e rebinding probabilities for filaments with shapes determined by 1, 2, 3, 5 or 10 modes. For this reason, we set the number of modes to 2 for all other simulations. Consistent with expectations, we found that th e probability of rebinding increased with the final filament length (Figure 34A). We looked at th ree filament lengths representing values similar to the minimum (1.6 m), the average (3.2 m), and the maximum (6.4 m) lengths from our experiments. Filaments with angles less than 70 had a ch ance of rebinding if they were allowed to elongate to 6.4 m, and filaments with angles less than 30 rebound to the track 100% of the time. Similarly, filaments that were allowed to elongate 3.2 m and 1.6 m had a probability of rebinding to the tracks at angles of incidence less than 40 and 30, respectively. Every filament that elongated past the track at an angle of 10 or less and was allowed to elongate 3.2 m, rebound to the track. Each fila ment length had the same decreasing probability trend with increasing angle of incidence, but the final length determined at what angles the decrease of probability began and ended. Binding probabilities were similar for the 60 at 6.4 m, 40 at 3.2 m, and 30 at 1.6 m fila ment lengths as well as at 40, 20, and 10, respectively.

PAGE 69

69 We also combined all of the data for all three filament lengths, which contained 300 filaments for each length, creating an average filament length of 3.7 m for 900 total filaments (Figure 3-4B). The trend and values for the comb ined data is very similar to that found in our experiments. Experimentally, no filament over 60 was found to rebind. In our simulations, that value was one iteration higher at 70, most likely due to the larger number of longer filaments in the simulations. Variations in the bindin g probability constant, Kp, as expected, had somewhat of an effect on the rebinding probability (Figure 3-5). Constants of 5 and 1 m-1sec-1 had similar results, indicating that the satu ration of the NEM-myosin may occur around a Kp of 1 m-1sec-1. In both cases, these Kp values allowed for rebinding to occu r at angles less than 60, with all filaments less than 30 rebinding every time. A Kp value of 0.5 m-1sec-1 resulted in similar rebinding probabilities at 5 and 1 m-1sec-1, with very slight diffe rences (no filaments rebound between 50 and 60, and a few filaments did not rebind between 20 and 30). Filament binding probability constants of 0.1 and 0.05 m-1sec-1 had a similar effect as the other three values up to 20 to 30, at which point the rebinding probability was significantly lower than the rebinding probability for the higher Kp values. The rebind ing probability of 0.05 m-1sec-1 also had a significantly lower rebinding probability than 0.1 m-1sec-1 in this range. All filaments, regardless of their Kp value, had a 100% chance of rebinding if the angle was less than 10. Finally, we looked at the eff ect of the persistence length of the filaments on the rebinding probability (Figure 3-6). As expe cted, the rebinding probability decreased as the persistence length increased, indi cating that the rebinding probability is directly affected by the stiffness of the filament.

PAGE 70

70 3.3.3 Alignment of Filaments In our next set of simulations, we varied th e track width, allowing the filaments to be initially placed in a random position within th e track. Similar to our experimental results, increasing the track width decreased the alignment of the filaments (Figure 3-7). We once again varied the number of modes to make sure that two modes were sufficient. We found that the number of modes had no effect on the alignment of the filaments as a function of track width (Figure 3-7A). Figure 3-7B show s that filaments within 1.5 m of the track edge have a higher average alignment than the averag e alignment of all filaments. This is in agreement with our experimental findings (Figure 2-8B). This alignment does decrease slightly with track width due to a combination of effects. First, the same number of filaments were tested for each track width (n = 1000) and their initial positions were evenly distributed within the track Therefore, the fraction of filaments with initial positions within 1.5 m of the edge decrea ses as the track width incr eases. In addition, as the track width increases, filaments that elongate to 1.5 m within the edge must have decreasing alignments to reach the edge with lengths between 1 and 16 m. Filaments must have a component perpendicular to the track edge, and th e only way for filaments to reach the edge as the track width increases without increasing the final le ngth is to decrease their alignment with the edge of the track. Once again, these resu lts show no dependence of alignment on the number of modes. Next, we varied the bindi ng probability constant, Kp, between values of 0.001 and 1 m-1sec-1 and found no dependence on the overall tre nd or values of the alignment as a function of track width (Figure 3-8). This indicated that Kp did not have an affect on the alignment at these values, which may corres pond to our experimental values above 0.5 M NEM-myosin (Figure 2-11A). Furthermore, th e actual persistence lengt h of bound filaments did

PAGE 71

71 not depend on Kp, with estimated values ranging from 13. 3 to 14.9 m with no apparent trend. These values were comparable to the persistenc e length calculated for the same discretization resolution with no binding (14 m), further indicating that binding did not affect the persistence of bound filaments. We next looked at even lower values of Kp (0.0005 and 0.0001 m-1sec-1) for a track width of 4 m to compare to our experimental analysis (Figure 3-9) We found that the alignment began to decrease for these Kp values. The difference between the alignment for Kp values of 0.001 and 0.0001 m-1sec-1 is comparable to the diffe rence between the values of alignment for 0.1 and 0.5 M NEM-myosin in Figure 2-8A. However, in our simulations, filaments were more aligned than in our experiments. We also observed a decrease in alignment at higher Kp values (0.01, 0.1 and 1 m-1sec-1). At these Kp values, the probability of rebinding to the tracks is high, however, once bound, the filaments may bi nd too often to fluctuate and realign with the track edge, causing the alignment to decrease. Experimentally, we may not have seen binding probabilities this high. In addi tion, we may have reached surface saturation, a factor that was not taken into account in our simulations. We also determined that the persistence length of the filament (Figure 3-10) had little effect on the alignment of filaments as a function of track width. Sli ght differences between the alignments of filaments with varying persistence lengths at lower track widths allude to a decrease in alignment with an increase in persistence length. 3.4 Discussion Our simulations have aided in interpreting ou r experimental results and in confirming the microcontact printing as a means to guiding elongating actin filaments. The simulations also provide a useful tool for designing patterns and experimental c onditions for optimal filament guidance. Furthermore, our simulation can be ap plied to other types of filaments, bundles of

PAGE 72

72 filaments, and filaments with attached particle s. We have replicated the experiments from Chapter 2 and have determined further paramete rs and limitations of the system. Filament lengths, NEM-myosin surface density, and persis tence length, in addition to the angle of incidence, affect the probability of a fila ment rebinding to the edge of the track. Specifically, longer filaments ha ve a higher probability of rebinding and also rebind with higher angles of incidence. As we expected through our mechanism based on experimental data and as our simulation confirmed, as the filament increases in length (passed the track edge), its probability of rebinding, which is based on length, increases. For this reason, in applications, it is important to control the length of the filame nts. With fewer filaments, each with greater length, alignment may be increased. Also, as expected, the higher binding probabi lity constants cause the rebinding probability to increase, but only within a ce rtain range of incidence angles. As found with our experiments, there is a surface saturation point, and we found this in our simulations that recreated the rebinding probability, with little difference between 0.5, 1 and 5 m-1sec-1. This lack of effect on filament alignment suggests that the angle of incide nce and the final filament length have a stronger effect on the probability of rebinding than the density of molecule s on the surface. If a filament leaves the track at an an gle greater than 70, the persistence length of the filament limits the filaments ability to bend enough to sample the NEM-myosin track surface. If a filament is too short, it also may not have enough energy to bend back to the track. Figure 3-5 shows that the only angles of in cidences that are affected by the binding probability constant were in the range between 20 and 40 (persistence length = 10 m, total filament length = 3.2 m). This small range of angles may explain why the binding probability had little effect on the overall al ignment of filaments evenly di stributed over the entire track

PAGE 73

73 width. These results support our mechanism propos ed in Chapter 2, indica ting that the filament thermal undulations are responsible for the alignment of the filaments on binding tracks, and that only very low surface densities will affect the re binding and alignment of filaments. Figure 3-9 demonstrates that at Kp values on the order of 10-4 m-1sec-1, small effects are seen on the overall alignment of the filaments. We were also able to vary the persisten ce length of filaments and study the effect on filament alignment. Filaments with longer persistence lengths we re less likely to rebind at higher angles due to the decrease in the magnit ude of their fluctuations. However, filaments with longer persistence lengths had slightly higher overall alignments on track s with smaller widths (< 10 m ). Therefore, in applications with filaments for longer persistence lengths, a filament with a small initial angl e will elongate with litt le changes in the alignment. However, if this filament leaves the track edge, it has less of a chance of rebinding to the track. Filaments with short persistence lengths may not align with the track edge, but may follow the track by continuing to cross the track edge and rebind, even though elongation itself on the track is less aligned. We also determined that the surface does not affect the persistence length of bound filaments, indicating that the su rface density did not alter the fila ment shape. This result is consistent with the experimental observation that the estimated measured persistence length of bound filaments did not vary significantly with the myosin density ( = 21.3 m for 0.1 M NEM-myosin, 15.7 m for 0.5 M, 14.6 m for 1 M, and 17.2 m, for 2 M). Therefore, we assume that the NEM-myosin surf aces lock the filaments into sh apes that are generated by the thermal fluctuations of the filaments themselves.

PAGE 74

74 Differences between experimental results and simulated results may be explained by the lack of certain conditions in the simulation. One of these factors includes the interaction between filaments, which we have not taken into account. Our simulations test one filament on a track, whereas our experiments contained many filaments per track. Experimentally, we eliminated surfaces that contained a high density of actin filaments. However, we could not completely eliminate filament interactions. Filaments may block each other from following paths that may be predicted for a sing le filament elongating on a track. In addition, we did not account for any variations in the tr acks of the filaments, including the possibility of diffusion of NEM-myosin monome rs from the printed re gion of the surface to the unprinted regions. Binding of filaments by these diffused NEM-myosin molecules may cause filaments that would have normally bent back to rebind to the tracks to remain in the unpatterned region, therefore decreasing the re binding probability. In our simulations, we assumed evenly distributed initial binding positions of filaments within the entire track, as well as evenly distributed initial filament angles. Experimentally, microcontact printing may cause slight variations of NEM-myosin surface density or activity, causing a less even distribution of filaments.

PAGE 75

75 Figure 3-1. The xand y-positi ons of elongating filament ends as a function of time. A) A filament leaving the track edge at a small angle eventually rebinds, as indicated by the decrease in fluctuations around 250 seconds. The initial angle of this filament was 19. B) The final instantaneous shape of the 3.2-m long actin filament from A. The thick blue line is the filament, the red circles represent binding of the filament to the surface, and the light black line at y = 5 is the track edge. C) A filament leaving the track at a large angle does not rebind. Fl uctuations are larger in the x-direction because the filament is elongating almost parallel to the y-axis. The initial angle of this filament was 79. Fluctuations continue to increase with time. D) The final shape of the 3.2-m long actin filament from C. For both filaments, the initial position was set to x = 0 and y = 4.99 (Kp = 0.1 m-1sec-1, n = 2, = 10 m).

PAGE 76

76 Figure 3-1. Continued

PAGE 77

77 Figure 3-2. Effect of change in length (s tep-size of simulation) on the filament rebinding probability. Results were approximately the same for both values of s, indicating that the step-size has little e ffect on the rebinding probability (Kp = 0.1 m-1sec-1, n = 2, = 10 m, total length = 3.2 m). Note that for s = 0.01 m, t = 1 sec, and for s = 0.0005 m, t = 0.5 sec, therefore keepi ng the elongation rate of the filaments constant.

PAGE 78

78 Figure 3-3. Effect of the number of modes on filament rebinding probability. For each mode, 300 filaments were analyzed. The trend and values for all modes were similar, indicating little effect of the number of modes on the rebinding probability (Kp = 0.1 m-1sec-1, = 10 m, total length = 3.2 m).

PAGE 79

79 Figure 3-4. Effect of total filament length on f ilament rebinding probability. A) For each length, 300 filaments were analyzed. The filament a ngles refer to the initial angles assigned to each filament at the start of the simula tion, which correspond to the angle at which the filaments crossed the track edge, or th e angle of incidence. The trend for all lengths are the same, however, the rebinding probability of the longer filaments began to decrease at a higher angle of incidence. B) The filament lengths from A were combined for an average filament length of 3.7 m (Kp = 0.1 m-1sec-1, n = 2, = 10 m). .

PAGE 80

80 Figure 3-5. Effect of binding probability constant, Kp (m-1sec-1), on filament rebinding probability. For each Kp value (0.05, 0.1, 0.5, 1, and 5 m-1sec-1), 300 filaments were analyzed. The filament rebinding probability was unaffected by the binding probability constant except for filaments cr ossing the track edge between 20 and 40. Within this range, the higher Kp values (0.5 1 and 5 m-1sec-1) had similar rebinding probabilities, while the rebinding probability decreased for both Kp values of 0.1 and 0.05 m-1sec-1 (n = 2, = 10 m, total length = 3.2 m).

PAGE 81

81 Figure 3-6. Effect of persistence length on filament rebinding probability. For each 300 filaments were analyzed. As the persistence length increased, the filament rebinding probability decreased at angles above 10 (Kp = 0.1 m-1sec-1, n = 2, total length = 3.2 m).

PAGE 82

82 Figure 3-7. Effect of track wi dth and number of modes on the a lignment of filaments. A) The simulation produced the same trend found e xperimentally, with no dependence on the number of modes. B) Effect of the track edge on the alignment of filaments. Only filaments or parts of filaments within 1.5 m of the edge of the track were included in the calculation of the alignment. The values are mostly within the error bars of the experimental data, with a slight decrease in alignment as the track width increases. The number of modes did not effect the alignment of filaments.

PAGE 83

83 Figure 3-8. Effect of track widt h and binding probability constant on the alignment of filaments. A) The simulation produced the same tr end found experimentally, with little dependence of alignment on the bi nding probability constant, Kp. B) Effect of the track edge on the alignment of filaments. Only filaments or parts of filaments within 1.5 m of the edge of the track were include d in the calculation of the alignment. The results are similar to 3-7A, with little dependence of the alignment on Kp (n = 2, = 10 m).

PAGE 84

84 Figure 3-9. Effect of binding probability on th e alignment of filaments. The alignment of filaments begins to decrease at binding probabilities with magnitudes of 10-4 m-1sec-1. This implies that not enough bind ing occurs to rebind filaments once they leave the edge of the trac k. The decrease at higher Kp values may indicate too much binding, resulting in a decrease of filament fluctuations and the subsequent decrease of alignment (track width = 4 m, n = 2, = 10 m).

PAGE 85

85 Figure 3-10. Effect of track width and persistence length on the a lignment of filaments. A) The simulation produced the same trend found e xperimentally, with a slight dependence of the alignment on the persistence length. The higher persistence lengths produced lower alignment values at narrower track widths (< 10 m). At a track width of 12 m, all three conditions produced the same ali gnment. B) Effect of the track edge on the alignment of filaments. Only filaments or parts of filaments within 1.5 m of the edge of the track were included in the cal culation of the alignment. Results are similar to Figures 3-7B and 3-8 B.

PAGE 86

86 CHAPTER 4 ACTIN-BASED MOTILITY OF LISTERIA AND PARTICLES ON MODIFIED SURFACES 4.1 Introduction We have replicated actin-based motility with both Listeria and ActA-coated 500-nm diameter polystyrene beads in bovine brain cell extract. NEM-m yosin treated glass coverslips were used to provide an anchor for the actin ro cket tails attached to the motile particles. Although actin rocket tails c ontained many filaments (~ 103), providing a system much different than single filament elongation, these surfaces were able to confine tails and control the propulsion of the associated particles, as observed through both real-time phase-contrast imaging and TIRF. We found that the paths of par ticles attached to c onfined actin-rocket tails lacked large thermal undulations (Brownian motion) relative to those particles and tails in solution. The effects of the NEM-myosin surfaces on the persistence a nd elongation rate of the actin rocket tail and the propulsion of the attached particle have been analyzed. Tr eatments of glass coverslips to create patterns or channels, including microcontact printing a nd photolithographi c lift-off of cyclic transparent optical polymer (CYTOP), a fully fluorinated polymer that reduces nonspecific protein interactions (110 ), were used to attempt to guide the path of the particles near the surface. 4.2 Materials and Methods 4.2.1 Listeria monocytogenes Growth and Protein Purification Listeria monocytogenes Strain Lut12 (pactA) over expres sing ActA (87) and labeled with 4,6-diamidino-2-phenylindole ( DAPI), was generously cultured and prepared by Will Zeile (University of Florida, Gaines ville, FL). Listeria strain s SLCC 5764 and DPL2823 were a kind gift of Dr. Daniel Portnoy (Univers ity of California, Berkley, CA).

PAGE 87

87 A truncated version of the transm embrane protein ActA with a His6 tag on the N-terminus was purified from Listeria monocytogenes (strain DPL2723) (111). Agar plates (containing brain heart infusion (BHI) media wi th agar and chloramphenicol) were streaked with bacteria, incubated for 24 hr at 37 C, and stored at 4 C. A colony of bacteria fr om the plates was added to 50 mL of BHI me dia diluted in ddH2O with 10 g/mL of chlora mphenicol and incubated overnight at 37 C while shaking at 225 rpm. Th e bacteria solution was then transferred to 1 L BHI with 10 g/mL of chloramphe nicol and incubated at 37 C wh ile shaking (180 rpm) for 6 to 8 hr (final OD600 = 0.6 to 0.8). The bacteria solution was cooled on ice fo r 20 min and clarified by centrifugation at 2700 g for 20 min at 4 C in a JA-10 rotor (Beckman Coulter, Inc., Fullerton, CA). Ammonium sulfate was slowly a dded and dissolved (1hr) in the bacteria solution for a final concentration of 50% (313.5 g/L) at 4 C and was left in 4 C for 3 hr while stirring to equilibrate. The precipitate was centrifuged at 4 C for 30 min at 7000 g in a JA-10 rotor and resuspended in less than 10 mL of Hepes-KCl buffer (HKB; 20 mM Hepes pH 7.4, 50 mM KCl,) supplemented with protease inhibitors (1 comp lete mini, EDTA-free ta blet per 10 mL, 1 mM PMSF) per liter of supernatant. The protein wa s eluted from solution using a 5 mL bed volume of Talon Ni-NTA resin equilibrated in HKB supplemented with 250 mM Imidazole. The fractions were dialyzed against HKB overnight to remove Imidazole from the buffer (111). Actin was purified and labele d with Oregon green 488 carboxy lic acid, succinimidyl ester (Invitrogen, Molecular Probes, Carl sbad, CA), with the same procedure described in Chapter 2. NEM-myosin was also prepared as described in Chapter 2. Frozen whole bovine brains were grinded into a fine powder in liquid nitrogen and mixed with an equal volume of sonication buffe r (10 mM Tris-HCl, pH 7.5, 2 mM MgCl2)

PAGE 88

88 supplemented with 10 g/mL each of pepstatin A, leupeptin, chymostatin, and 1 mM PMSF. The mixture was dounce homogenized 30 on ice, sonicated on ice with a tip sonicator for 30 sec bursts at 25% power, and then centrifuged at 17,000 g for 20 min at 4 C in a Ti60 rotor (Beckman Coulter, Inc., Fullerton, CA). Th e supernatant was then centrifuged at 118,000 g for 1 hr at 4 C in a Ti60 rotor. The supernatan t was recovered and supplemented with 1 mM DTT and 1 mM ATP. The extract was aliquoted, flas h frozen with liquid ni trogen, and stored at -70 C (112). 4.2.2 Bead Preparation 500-nm diameter polystyrene beads (Polyscien ces, Inc., Warrington, PA) were diluted in MOPS buffer (100 mM MOPS pH 7.0 with KOH) for a final of 0.5% solids solution. The beads were rinsed twice by centrifuging at 16,000 g for 5 min at 4 C (Eppendorf 5415C microcentrifuge), and resuspending in MOPS buffer. Bis[sulfosuccinimidyl] suberate (BS3) (Pierce, Rockford, IL) was diluted to 20 mM and added dropwise to the beads, and the solution was incubated for 15 min at room te mperature while shaking. The BS3 solution was removed by pelleting the beads through centrifugation (5 min, 16,000 g) and ActA was added directly to the beads at ~ 200 to 250 g/mL. The solution in cubated at room temperature for 1 hr while shaking gently. The ActA solution was removed by centrifugation (5 min, 16,000 g), and the beads were resuspended and rinsed twice in a 20 0 mM solution of glycin e methyl ester in MOPS buffer to block all remaining BS3 binding sites. Beads were rinsed once in MOPS buffer, resuspended in brain extract buffer (20 mM Hepes, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 100 mM KCl, 0.2 mM CaCl2, 150 mM sucrose), and stored at 4 C until activity of the beads decreased significantly, which typically occurred within one week (William Zeile, unpublished).

PAGE 89

89 4.2.3 Motility Assay Motility assays consisting of extract from bovine brain cells were used to mimic in vivo conditions for actin-based motility of Listeria and 500-nm ActA-coated pol ystyrene bead. An aliquot of bovine brain extract was thawed and clarifie d by centrifugation at 16,000 g for 10 min at 4 C. The supernatant was aspirate d and supplemented for a final composition (by volume) of 10% creatine kinase (CK)-ATP regenera ting solution (7.5 mM creatine phosphate, 2 mM ATP, 2 mM EGTA, 2 mM MgCl2, 50 g/mL creatine kinase), 10% protease inhibitors (10 g/mL each of pepstatin-A, chymostatin, and leupeptin), 10% (10 mM) dithiothreitol (DTT) and 75% clarified extract (113). After incubating on ice for 5 min, Oregon gr een 488-labeled actin wa s added to a final concentration of 5.7 M a nd either Listeria (final concentratio n = 1 to 1000 dilution of stock) or ActA-modified beads (final concen tration = 1 in 100 to 1 to 400 d ilution of stock) were added in the final extract. Actin tails formed on these be ads within 30 min and pr opelled the particles in solution. Motility assays used on modified surf aces were incubated in a microcentrifuge tube until approximately 15 to 30 min after the solution was first mixed. This typically allowed enough time for short rocket tails to form on the beads, which could then bind to the surface. Flow-cells with cleaned gla ss coverslips were treated with either 2 M NEM-myosin diluted in myosin dialysis buffer or 1% BSA in TM buffer (10 mM Tris, pH 7.5, 2 mM MgCl2) for 1 min, followed by 1% BSA in TM buffer to wash any unbound NEM-myosin molecules and to passivate any exposed glass surface. A motility assay containing Listeria or ActA-modified beads was added immediately after the BSA rinse. 4.2.4 Fabrication of Channel Devices Using a photolithographic lift-off technique, CY TOP was patterned on glass coverslips. A glass coverslip was photopatterned with two-coatings of NFR016, a negative photoresist. Using

PAGE 90

90 an e-beam evaporator, a 50 -thick chrome layer was deposited on the top surface of the photoresist-patterned coverslip. CYTOP was th en spin-coated onto the surface at 4000 rpm for 20 sec and was cured at 100 C for 10 min. Th e slide was soaked in acetone for 5 min and sonicated briefly at low power. This caused the regions with the photoresist layer to lift off the surface, exposing the glass. The CYTOP layer was found to be approximately 2 m thick based on measurements from an Alpha-Step IQ profil omoter. PDMS stamps and CYTOP patterned surfaces were prepared by Dr. Suzanne Ciftan-H ens (International Technology Center, Raleigh, NC). 4.2.5 Microscopy and Analysis Samples were observed using a Nikon Diaphot inverted photomicroscope equipped with phase-contrast optics for up to 2.5 hr after motility assays were mixed. Images were acquired using a digital CCD camera and analyzed usi ng MetaMorph software. Time-lapse sequences were 5 to 10 min long with images acquired at 15 sec intervals. Objective-based TIRF (as described in Chapter 2) was used to image de tails on actin rocket tails bound to the surfaces approximately 2 to 4 hr after motility assays were mixed. Random fields-of-view were ta ken for three experiments and combined to determine the fraction of bound actin rocket tails on BSA and NEM-myosin-treated surfaces. We only included those tails that were almost completely in the field-of-view for approximately the entire time-lapse sequence of 5 min. Particles and tails that were not attach ed to the surface were distinguished from those that were attached beca use of their larger fluc tuations (which also hindered our measurements of tail length and bead velocity). A set of tails were considered to be partially attached; they were either attached at part of the tail th at was not near the bead (causing a noisy bead trajectory) or the bead was attached to the surface but was not motile. Particle paths were manually tracked over time using MetaMorph software.

PAGE 91

91 The elongation of the actin rocket tails was cal culated by measuring the change in length of the actin rocket tail near the particle at a minimum of three time points for each tail. The average elongation rate was calculated by pooling all of the data within each surface treatment and determining the slope of a best-fit line (with intercept set to 0) for the change in length versus the change in time. Measurements of tails that were not bound to the surface may have been skewed due to parts of the tail leaving and re-entering the field-of-v iew. This limited our data of tails on passivated surfaces for the final analysis. Finally, tails were traced in the last frame of every time-lapse for both BSA and NEM-m yosin treated surfaces to determine if the NEM myosin surface had any effect on the persis tence of the tail. The distance between the end-points of the tails was square d and compared to the total length of the tail. 4.3 Results 4.3.1 Confining Particle Propulsion to the Surface Actin-based motility was induced in vitro in cell extracts using the motility assay described above. Both Listeria and ActA-coated 500-nm diameter polys tyrene beads produced actin rocket tails, as shown in images taken using phase-con trast microscopy (Figure 4-1). The actin tails looked identical in both cases, despite the slight difference in size and shape between the Listeria and the bead. The rocket tails elongated in solutio n, and in addition, rota ted in three dimensions over time (Figure 4-2). The ro cket tails took on a variety of different conformations, many tending to have a curved or helical shape. In order to manipulate the path of the particles, we eliminated the rotation and thermal motion of the rocket tails in solution by confining them to NEM-myosin glass coverslips. Actin tails that were attached to the surface were distinguished from tails that were not attached by their decreased Brownian motion observed in real-time phase-cont rast microscopy. Figure 4-3 shows an actin rocket tail attached to Listeria on an NEM-myosin surface over time. The rocket

PAGE 92

92 tail remained stationary while the bacterium wa s propelled forward. On a BSA-coated surface, the actin rocket tail elongated but also change d position over time. Figure 4-4 shows similar results for a 500-nm diameter bead attached to a tail on an NEM-myosin and BSA surface. In 4-3B and 4-4B, the tail and the Listeria or bead remained in the focal plane for the entire time-lapse, moving only in the xand y-dire ctions. Many free tails also moved in the z-direction, coming into or out of the focal plane during the time-lapse. In addition, some free tails had part of their structure in the focal plane and the remainder above the focal plane for the entire sample time. 4.3.2 Effectiveness of NEM-Myosin Surfaces Many samples contained a high percentage of actin rocket tails bound to the surface, as indicated by their lack of movement over time (Figure 4-5). The number of tails bound per field-of-view varied, depending on the sample, th e time of the assay, and the position in the flow-cell. On NEM-myosin-coated surfaces, equal fractions of actin rock et tails attached to 500-nm diameter beads near the surface were completely bound or partially bound, comprising ~60% of the total number of tails (Figure 4-6). Partial binding occu rred when a tail was attached at certain points away from the particle or wh en a particle attached to the surface was not moving. On the BSA surfaces, only one tail was attached to the surface, indicating that the binding of the actin rocket tails was caused by specific interactions with NEM-myosin. The movement of the free tails in solution cause d the trajectories of the attached beads to appear chaotic compared to the smooth trajecto ries of the particles attached to bound tails (Figure 4-7). Particle motility in the bound case was likely controlled by actin filament elongation within the actin rocket tail. For particles not bou nd (on either NEM-myosin or BSA-treated surfaces), the trajectory was chaoti c because, in addition to elongation, the rocket tail itself was moving with respect to time. These results suggest that NEM-myosin can

PAGE 93

93 effectively bind actin filaments within a large actin tail, confining the tail to the surface for some time while continuing to propel particles attached to the actin rocket tail in a more controlled manner. In some cases, the interaction between the NEM-myosin and the actin rocket tail was enough to confine actin rocket tails with helical shapes, as shown in Figure 4-8. It is unknown in this case if the actin rocket ta il landed on the surface as a helix or elongated into that shape while bound to the surface. However, the particle did c ontinue to follow the curvature of the helical path as its tail elongated, which is most likely an effect of th e long persistence length of the rocket tail. Even though the entire tail remains in the same position over time for many samples, it is likely that only some regions of the tail were attached. TIRF images show that regions of the rocket tails were clearly bound to the coverslips (Figure 4-9), while other regions were not interacting with the surf ace. Slightly above TIRF range (> 2 00 nm from the surf ace), the entire rocket tail was observed, proving that, in ma ny cases, the actin rocket tail was bound randomly along its length. This implies that the entire length of the tail did not need to be interacting with the surface for the tail to remain bound. An en larged TIRF image of the actin rocket tail revealed potential actin-surface inte ractions at the sides of the tail, where single filaments or small bundles seemed to protrude away from the ma in structure (Figure 4-10) It is unclear if these single filament protrusions were located at the bottom of the tail as well. The binding interactions may have occurred mainly with these filaments. 4.3.3 Particle Velocity and Tail Characterization We next looked at the effect NEM-myosin substrata had on both elongation rate and persistence of actin ro cket tails. Figure 4-11 shows a hi stogram of tail elongation rates on both NEM-myosin and BSA surfaces. NEM-myosin surfaces had a narrower distribution of instantaneous rates, whereas the BSA surfaces were more wide-spread, containing some rates

PAGE 94

94 below 0 m/sec. The change in length versus time was plotted for bound actin rocket tails on NEM-myosin surfaces and free tails on BSA surfaces (Figure 4-12). The slope was calculated from a best-fit line to determine an ove rall tail elongation rate of 0.0157 0.0005 m/second for tails attached to NEM-myosin surfaces and 0.0135 0.001 m/second for free tails near a BSA surface, showing little effect of the surface on the rate of particle propulsion. Accurate measurements were difficult to obtain for the free ta ils due to the motion of the tails over time in the xand y-direction and also in and out of the focal plane. This difficulty in measurement could imply that the free fila ment elongation rate reported ma y be lower than the actual elongation rate due to loss of data. This ma y also account for the wider distribution of elongation rates and the negative elongation rates acquired for certain samples. The persistence of the actin ro cket tails was measured by comp aring the total length of the tail to the distance between the st art and the end of the tail. The ratio of distance to length of the tails bound to the NEM-myosin surface was similar to that of free tails up until lengths of approximately 35 m, at which poi nt bound or partially bound tails have a slightly higher ratio than free tails on NEM-myosin or BSA surfaces (Figure 4-13). This implies that the NEM-myosin had a slight effect on the final shap e of the actin rocket tail and subsequently, a slight effect on the final path of the 500-nm particles. Furthe rmore, the average length of the actin rocket tails measured on the NEM-myosin surface (28.4 1.5 m) was statistically greater than the average length for those on the BSA surfaces (21.8 1.7 m ), with a two-tailed p-value = 0.0196. This result indicates that ta ils bound to NEM-myosin-coated substrata contain more tail length within a two-dimensional plane than free tails. In this case, the NEM-myosin surface had a clear effect on the tail shape, eliminating the freedom of the tails to bend into the z-direction.

PAGE 95

95 4.3.4 Guiding Particle Propulsion Microcontact printing was used to attempt to further guide the particles, similar to the guidance of single filaments. There are many differences between actin rocket tails and single filaments, including size and solu tion conditions, indicating that this method may not be as effective for the large bundles of actin filaments. Initial attempts show selective binding of the particles to the NEM-myosin, but confinem ent does not compare to the tails bound to unpatterned NEM-myosin surface (Figure 4-14). Although tails remained near the surface, many rotated or moved around one point of attachment, implying that short ta ils shown in the time series were only partially bound to the surface. Furthermore, these tails did not elongate over time, preventing motility of the attached beads. Factors that may be inhibiting the actin tail-NEM-myosin interaction on the microcontact-printed surfaces include the density of the NEM-myosin layer, conformation of NEM-myosin molecules and the bi nding activity of the NEM-myosin with actin filaments. In addition, the brain extract used contains large amounts of protein, including myosins, tropomyosins, and actin. All of these proteins may have bound to the regions of NEM-myosin non-specifically, blocking the binding of the actin rocket tails to the surface. Three-dimensional surface structures using patterned CYTOP on glass coverslips have been tested for their ability to guide 500-nm diameter beads undergoing actin-based motility. While many actin tails were confined to the NEM-myosin-treated glass portion of the surfaces of these devices (Figure 4-15), the ability of the device to guide the par ticles has not yet been determined. Figure 4-16 shows a particle being propelled by an actin rocket tail encountering a wall at a perpendicular angle. The particle was propelled over the wall, indicating that the height and design of the wall were not optimal to guide the path of the particle.

PAGE 96

96 4.4 Discussion 4.4.1 Mechanics of Actin Rocket Tails on Surfaces The results obtained in this chapter suggest that surface manipulation may be used to confine the propulsion of both Listeria and 500-nm diameter partic les undergoing actin-based motility. NEM-myosin adsorbed to glass surfaces ha s the ability to confine the actin rocket tails to the substratum while continuing to propel the objects. NEM-myosin molecules bind to single actin filaments, thus the tails must be confin ed to the surface by ma ny interactions between single actin filaments within the tail and single NEM-myosin molecules. Based on our TIRF images, NEM-myosin may create a place for actin filaments protruding and elongating from the sides of actin rocket tails to bind. In solution, filaments are likely protruding from all sides of the tail. Therefore, the side of the tail facing the surface may be interacting with the su rface through these small filaments ra ther than filaments that are cross-linked within the main tail. These filame nts may have been side branches created by the presence of Arp2/3 in the extract (114 ). If these filaments are present in vivo, they may function by incorporating in the cytoskeletal actin netw ork, providing an anchor for the propulsion of Listeria in the cytoplasm. Our TIRF images also i ndicate that the entire length of the tail does not appear to be bound to the surface. Tails may be confined to the surface at random places along their length, and this may be sufficient fo r overall confinement of the tail. Another possibility is the areas that do not appear dire ctly on the surface may be cross-linked to the nonspecific actin filaments that are attached to the surface, creating a network from the NEM-myosin surface to the tail. Only filaments in the tails that are contacti ng the substratum are av ailable for binding to the substratum, a fact which limits the effect the substratum properties has on the entire actin tail and the propulsion of the particle. The most signifi cant effect of the surface-tail interaction is the

PAGE 97

97 ability to reduce the noise in particle trajectories. By controlling the tr ajectories, particles are more likely to follow a designated path on a surface and move against flow or diffusion gradients. The tail elongation rate and two-dimensional shape was not affected significantly by the NEM-myosin surfaces. (The surface did affect the shape of the tail with respect to the z-direction.) The rocket tails contain many filaments, all of wh ich contribute to the overall elongation rate and shape of the ta il. Since only filaments near th e surface are in teracting with NEM-myosin, it is expected the surface interactio ns will have little effect on these parameters. Furthermore, the filaments protruding from the si des of the rocket tail, which are likely to be interacting with the substratum, should not eff ect the elongation rate at all since they are not elongating near the surface of the motile object. 4.4.2 Biochemical Considerations Biochemical modifications may control cert ain parameters of the system, including percentage of beads that form tails, length of tails and velocity of the partic les. We have tested a variety of extracts, including rat brain, bovine brain, and human platelet extracts. All three of these extracts supported motility of Listeria and beads. A thorough analysis of the extracts may be beneficial for optimization of the assay. Th e extracts may contain variable amounts of VASP, Arp2/3, ATP and ADP, protease inhibitors, ADF, cofilin, profilin and actin, as well as many unknown proteins that could aid or inhibit certain aspects of actin-based motility (115). Cameron, et al. showed that extract dilution, in addition to bead diameter and ActA coverage, influenced the speed of particles and the tail cu rvature (116). We expect these parameters will have the same effect on beads attached to the surfaces. Another important factor is the time-sensitivit y of the 500-nm ActA-tre ated particles, i.e. the same particles may behave differently as so on as one day after initially making and testing the sample. The particles tend to aggregate and the surface concentration of ActA decreases over

PAGE 98

98 time through desorption. Even though ActA was cros s-linked to the surface of the particles, it is possible that some ActA only physic-adsorbed to the beads and is aiding in tail formation and elongation. The subsequent desorption of this ActA may be enough to d ecrease tail formation significantly. Surface binding is also dependent on the length of the tails when the motility assay is added to the modified glass coverslips. Before actin tails begin to form, an actin cloud, or a cross-linked actin network, surrounds the ActA-c oated motile object. The bead inside this network may make small fluctua tions, eventually causing the netw ork to break spontaneously to one side of the bead, i.e. symmetry break (117). If the assay is added to the surface during the actin cloud state, the cross-linked actin network may bind to the surface, trapping the bead inside the network, ultimately preventing symmetry breaking. All of these factors combined make the biochemistry of this system complex. It may be beneficial in the future to move from cell extracts to single component systems (115), whic h could enhance the effect of the surfaces on the actin rocket tails and particle propulsion as well as allow more control over the velocity and curvature of the particles. 4.4.3 Considerations for Bionanotechnology As mentioned before, each NEM-myosin molecule binds to only one filament in the tail, leaving most of the tail unaffected by this interact ion. This characteristic of actin tails could indicate that modified substrata alone may have a limited effect on controlling the trajectories of the 500 nm beads on the surface. With larger acti n structures propelling particles, a combination of channels and modified substr ata may provide the most contro l over the trajectory of the particles (105). Microcontact printing may be fu rther investigated as a way to confine filaments to the surface, but it is unlikely that this technique will provide the same advantages that it does for single actin filaments. The actin rocket ta il is much stiffer than a single actin filament,

PAGE 99

99 suggesting that the smaller thermal fluctuations of the tail would make it difficult to bend back and rebind to a track. In addition, the path and velocity of the propelled particle is dependent on many filaments that may not be interacting with the surface. For these reasons, physical barriers may be needed to provide contro l over the particles path. Further optimization of the hei ght of the channels may preven t the particles from traveling over the top of the channel walls. Other design s, such as undercuts may also prevent this, as shown to be effective by Hess, et al. for the guid ance of sliding microtubule s (17). If the channel walls are high enough, beads may be able to take paths that include sharp or rounded turns. In addition, directionality of the particles may be easily obtained through the use of paramagnetic particles or electric fields. By allowing tails to bind first and then applying a force, the particles may change their path toward a specified direction. Particle motility for potential use in nanodevices has been confined to methods using single filament microtub ule/kinesin or actin/myosin sliding assays, where particles are either attached to the filament or the motor protein (2 3, 6, 7). Bohm, et al. successfully showed motility of large, micron-sized particles of various materials by attaching the objects to kinesin and allowing them to move along an array of ali gned, isopolar microtubules (3). We have added another method of particle motility that expl oits actin polymerization and the ActA/VASP complex and can be useful in the future for nanodevices.

PAGE 100

100 Figure 4-1. Listeria and 500-nm diameter bead propelled by actin rocket tails. A) Listeria placed in cell extract formed actin rocket tails at its rear. B) Polystyrene 500-nm diameter particle treated w ith the surface protein ActA emulated the behavior of Listeria under the same conditions, forming actin ro cket tails. Images were taken in phase-contrast (scale bars = 5 m). Figure 4-2. Rotation of a helical actin rocket tail in solution. A polystyrene 500-nm diameter particle contained an actin rocket tail which rotated in th ree dimensions over time. The arrow indicates where the tail crosses its elf in the various views. The images were taken 1 minute apart in phase-contrast (scale bar = 5 m).

PAGE 101

101 Figure 4-3. Listeria rocket tails on NEM-myosin a nd BSA-treated surfaces. A) Listeria actin rocket tail is bound to an NEM-myosin tr eated surface. The tail did not move over time, as indicated by the top line. The actin rocket tail propelled the Listeria as seen by the increasing distance between the bacterium and the bottom line over time. Images shown were taken 75 seconds apart. B) Listeria actin rocket tail remained free in solution when exposed to a surface passivated with BSA, as indicated by the movement of the entire tail over the time lapse. Note there are two Listeria in this time-lapse sharing a membrane. Images s hown were taken every 45 seconds (scale bars = 5 m). Figure 4-4. 500-nm diameter beads attach ed to rocket tails bound on NEM-myosin and BSA-treated surfaces. A) Actin rocket tail is bound to an NEM-myosin treated surface. The tail did not move over time, as indicated by the top line. The actin rocket tail propelled the particle as seen by the increased distance between the particle and the bottom line. Images shown were taken 75 seconds apart. B) Actin rocket tail remained free near a surface pa ssivated with BSA, as indicated by the movement of the entire tail over the time lapse. Images shown were taken every 75 seconds (scale bars = 10 m).

PAGE 102

102 Figure 4-5. Fields-of-view with large percentage of tails bound to surface. A) Actin rocket tails on Listeria bound to an NEM-myosin surface. Many tails remained in position while elongating over the 2.5 minutes between im ages. B) Actin rocket tails on 500-nm diameter beads are also bound in a high perc entage to NEM-myosin treated surface. Tails remained in position between the first image and the second image, taken 3.5 minutes apart (scale bars = 20 m).

PAGE 103

103 Figure 4-6. Fraction of actin rocket tails bound to NEM-myosin and BSA-treated surfaces. Only one tail was bound to the BSA surface. Of the tails on the NEM-myosin surface, 28% were bound and successfully propelling a pa rticle, 31% were partially bound, and 41% were free in solution. Tails were analyzed from 20 randomly selected fields-of-view for each surface from three diffe rent experiments. Error bars represent standard error = (f(1-f)/N)1/2, for N total filaments.

PAGE 104

104 Figure 4-7. Change in xand y-position over time for beads on NEM-myosin and BSA surfaces. A) A 500-nm diameter bead attached to an actin rocket tail bound to an NEM-myosin coated surface is propelled forward in a smooth trajectory. B) A 500-nm diameter bead attached to an actin rocket tail th at is in solution near a BSA-coated surface underwent Brownian motion as indicated by the noise in the position over time. Figure 4-8. Helical actin rocket tail confined to NEM-myosin-t reated surface. Images were taken 1 minute 15 seconds apart (scale bar = 5 m).

PAGE 105

105 Figure 4-9. Actin rocket tails in total internal reflection fluo rescence microscopy. A) Actin rocket tail attached to Listeria was bound to the NEM-myosin surface, as shown in TIRF. The actin rocket ta il appears to have smaller bundles or single filaments extending off the side of the main structure. B) Image of A taken in fluorescence just above the 200-nm TIRF region, showing the entire rocket tail. Only a small portion of the length of the tail was bound to the surf ace. C) Actin rocket tail attached to a 500-nm diameter bead was bound to a NEM-myosin surface. The arrow points to where the bead was most likely located. D) Fluorescent image slightly above TIRF corresponding to C (scale bars = 10 m).

PAGE 106

106 Figure 4-10. Magnified image of ac tin rocket tail with protruding filaments. The actin tail had single filaments or small bundles of actin extending from the main structure. Short filaments that may have formed spontane ously in solution were also bound to the surface (scale bar = 5 m).

PAGE 107

107 Figure 4-11. Actin tail elongation on NEM-m yosin and BSA surfaces. A) Histogram of instantaneous tail elongati on rates (frequency) observe d for actin tails bound to NEM-myosin surfaces. The peak occurs between 0.015 and 0.016 m/sec (n = 280, 80 filaments). B) Histogram of instanta neous tail elongation ra tes observed for free actin tails near a passivated BSA-treated glass surface. The peak occurs between 0.006 and 0.008 m/sec. The wider spread of rates, including negative elongation rates, was caused by difficulties in measuring the length of tails that were not attached to the surface and were potentially moving in and out of the focal plane (n = 169, 58 filaments).

PAGE 108

108 Figure 4-12. Average tail elongation rates determined from the slope of a be st-fit line. A) The changes in length of the tail s attached to NEM-myosin surfaces were plotted against time to determine an average rate of 0. 0157.0005 m/sec. The two outliers in the histogram (Figure 4-11A, > 0.05 m/sec) we re excluded from the plot (n = 280, 80 filaments). B) Values less than 0 and greater than 0.05 were eliminated (Figure 4-11B) to determine an average elonga tion rate of 0.0135.001 m/sec for free filaments near BSA-treated surfaces (n = 146, 50 filaments). An unpaired t-test gives a p-value of 0.04, indicating that the two elongation rates are significantly different.

PAGE 109

109 Figure 4-13. NEM-myosin surface effect on persiste nce of tail. Tail lengths were measured as they appeared in the two-dimensional focal plane. The total length of the filaments was typically longer than the distance betw een the end-points of the tail (r). Bound and partially bound tails (n = 84 and n = 40, respectively) between 35 and 60 m long seem to have slightly more persistence than free tails on NEM-myosin surfaces or BSA surfaces (n = 56 and n = 197, respectivel y). We were able to measure more length per filament for the bound tails due to their confinement in the focal plane.

PAGE 110

110 Figure 4-14. Stamped surfaces with 500-nm diameter beads attached to actin tails. Very faint lines in phase-contrast were present and ar e enhanced by the white dashed lines. Beads seemed to attach to the regions within th e track boundaries; however, elongation was not observed, and tails seemed to rotate or move slightly over time. Images shown were taken 75 seconds apart (scale bar = 10 m).

PAGE 111

111 Figure 4-15. Actin tails bound to NEM-myosin-tr eated exposed glass of fabricated device. Black regions in the images are CYTOP, while all other area is exposed glass. The arrow points to a 500-nm diameter bead that was propelled by an actin rocket tail bound to the glass. No tails were observed elongating inside th e channels. Images were taken 75 seconds apart (scale bar = 20 m).

PAGE 112

112 Figure 4-16. Actin rocket tail encounters CYTOP wa ll. A) Image of entire region of the device. The black region is CYTOP while all othe r regions are NEM-myosin treated glass (scale bar = 20 m). B) An actin rocket tail confin ed to the glass surface encountered the wall at a perpendicular angle and propell ed the particle over the channel wall, indicating that channel walls ma y need to be higher to gu ide the particles. Images shown are at 2.5 minute in tervals (scale bar = 5 m).

PAGE 113

113 CHAPTER 5 SINGLE FILAMENT ACTIN-BASED MOTILITY OF PARTICLES 5.1 Introduction The use of rocket tails for propulsion of larger particles and bacteria is challenging in that guiding stiff bundles of actin filaments on modified surfaces is difficult without three-dimensional barriers (channel walls). Smaller particles (50 nm in diameter) have potential to be propelled by a single filament or a fe w actin filaments while behaving similarly to elongating single filaments w ithout particles. As st ated earlier, this ma y lead to guidance of these particles using microcontactprinted tracks (Chapter 2). The challenge thus far has been observing elongating single filaments bound to particles while persistently attached via the ActA/VASP complex. The only previously published observation of a single filament on a 50-nm diameter ActA-coated bead was obtained using transmi ssion electron microscopy (TEM) (89). Recently, our group has observed many single filaments on 50-nm ActA-coated beads in TEM (Sturm, C., J. Phillips, W. Zeile, K. Interliggi, R. Dickinson, and D. Purich, unpublished), lending promise to the possibility of single-filament bead propulsi on for nanodevices. Processively elongating single filaments have been more frequently and easily observed using formins attached to 1-m diameter beads (27). A bead with a diamet er of 1 m would be too large to guide on microcontact-printed surfaces alone, but the a ttachment of single filaments through formins could be applied to smaller particles. With our recent experiments, we have shown that single filaments ar e attached at their (+)-ends to small ActA-coated beads, s upporting the end-tracking motor mechanism for ActA-induced filament elongation (Sturm, C., J. Phill ips, W. Zeile, K. Interliggi, R. Dickinson, and D. Purich, unpublished). This result also ve rifies the potential for guiding small particles

PAGE 114

114 with single filament elon gation through filament end-tracking mo tors. To address the possibility of single-filament actin-based motility in nanode vices, we first looked at attaching particles, rather than tails, to surfaces. This method decr eases the Brownian motion of the beads to give a reference point for actin filame nt-particle interacti on and allows dynamic observation in TIRF. Once filaments could be observed consistently, the system was optimized to visualize only a single filament or a few filaments per bead. Actin filaments were characterized by their fluorescent intensities to elucidat e the mechanism of attached filament elongation near the bead surface. Finally, attempts were made to at tach actin filaments under these conditions to NEM-myosin surfaces to observe a motile bead. The complexity of this system was assessed through these experiments. 5.2 Materials and Methods 5.2.1 Protein Preparations ActA, bovine brain extract, actin, and NEM-m yosin were all prepared as described in Chapter 2 and Chapter 4. 5.2.2 Bead Functionalization A 500-L aliquot of 50-nm diameter silica b eads (Polysciences, Inc., Warrington, PA) was pulse sonicated 20 at low power (Benson Sonifier 450) to break apart aggregates of beads and suspend them evenly in solution. The beads were incubated for 1 hr at room temperature with 0.2% gluteraldehyde with ge ntle shaking. The solution was centrifuged at 16,000 g for 5 min at room temperature followed by aspiration of the supernatant and resusp ension of the beads in 500 L of 1600 g/mL of ActA in brain extract buffer. The solution incubated for 40 min at room temperature with gentle shakin g, followed by centrifugation (16,000 g, 5 min, 4 C). The beads were resuspended in a 500 L solu tion of 3100 g/mL of Or egon green 488-labeled BSA and incubated for 1 hr at room temperatur e. The solution was centrifuged as before,

PAGE 115

115 resuspended in 500 L of 100 mM glycine methyl ester in brain extract buffer to block any reactive gluteraldehyde molecule s, and pulse sonicated. The solution was centrifuged and resuspended in brain extract buffer twice to wash away any excess protein or chemicals and then stored in brain extract buffer at 4 C. 5.2.3 Motility Assays Two motility assays were tested using the 50-nm diameter beads. First, beads were attached to the surface and exposed to a motility assay. The second assay bound actin filaments formed in a solution of ActA-coated 50-nm diam eter beads and extract to NEM-myosin surfaces, similar to the motility assays described in Chapter 4. 5.2.3.1 Attached beads Aminopropyltriethoxysilane (APES)-treated su rfaces were prepared as follows. Glass coverslips were rinsed in water followed by 100% ethanol. Dry coverslips were then incubated in a water-free acetone bath for at least 1 hr before transferring to a 2% APES solution in water-free acetone. Coverslips were incubated in the APES solution for two days, rinsed twice in acetone and once in water, and set out to dry. These coverslips were incubated in a 1% gluteraldehyde solution for 20 min at room temperature and rinsed twice in distilled water. The coverslips were dried and then made into flow channels using double-sided tape. The 50-nm diameter ActA-treated silica beads were diluted in brain extract buffer, added to the flow channel and incubated for 10 min at room temperature. A 100 mM solution of glycine methyl ester in brain ex tract buffer was flowed into to the channel at twice the chamber volume and incubated for 20 min, followed by brai n extract buffer for storage until use. After incubating on ice for 5 min, supplemented bovine brain extract (see Chapter 4) with either 100% or 60% extract diluted in brain extract buffer was mixed with 5 M of Oregon green 488-labeled actin and the solution was flowed into the channel. Actin filament structures were visible within

PAGE 116

116 a few minutes. When noted, reactions were fixe d with 1% gluteraldehyd e after the reaction was allowed to proceed for 1 to 5 min. 5.2.3.2 NEM-myosin surfaces Flow-cells with cleaned glass surfaces were treated with 2 M NEM-myosin for 1 min followed by 1% BSA in HS-TBS and 1% BSA in LS-TBS. A motility assay containing a 1 to 200 dilution of ActA-coated silic a beads was added to the surf aces. (See Section 4.2.4 for a description of the motility assay.) 5.2.4 Microscopy and Analysis All samples were observed using TIRF micr oscopy. MetaMorph was used to acquire images and to perform analysis. Fluorescent in tensity line-scans were performed on samples to determine the change in intensity over time at various positions on actin filaments. Rate constants for the recovery of fluorescence at these positions were calculated by fitting the data to Equation 5-1. kt Eqo Eqe II II (5-1) I is the intensity (arbitrary units) at time, t (sec), Io is the initial intensity immediately after photobleaching, IEq is the equilibrium intensity reached and k is the rate constant (sec-1). Microsoft Excel was used to solve for k and IEq by solving for the minimum difference between the experimental and theoretical values. 5.3 Results 5.3.1 Actin Asters We observed growth of actin filaments from 50-nm ActA-coated b eads covalently bound to glass coverslips using TIRF. These actin filame nts formed actin asters, or star-like structures, around the beads over time (Figure 5-1). The highe st intensity of fluores cence was at the center

PAGE 117

117 of the beads, and the filaments intensities seem ed to decrease with increasing distance from the bead (Figure 5-2). One explanation of this decrease implies that new monomers were incorporating at the particle (( +)-end elongation near th e bead) and that the older regions of the filament (the (-)-ends), which have been exposed to light in TIRF and we re therefore dimmer due to photobleaching, were further aw ay from the bead. Another po ssibility for the decrease in intensity is that shorter filame nts closer to the surface were di rectly below longer filaments, which were slightly above the surface. In either case, many of the filaments present demonstrated the same trend (Figure 5-2). To investigate monomer addition further, we photobleached fields-of-view by exposing the samples to the laser at its highest intensity a nd observing the recovery of the fluorescence. Figure 5-3 is an example of photobleaching wher e filaments reappeared on the particle and on the glass surface in the field-of-view. A closer look indicates that the reappearing filaments on the surface of the bead seem to correspond to a filament existing on the bead before photobleaching (Figure 5-4). No t all filaments present before photobleaching reappeared. The fluorescent intensity of two filaments wa s plotted against the filament distance from the center of the bead for four different time points after photobleaching occurred (Figure 5-5). In the first case, the filament recovered, followi ng the same trend of intensity along the length of the filament until approximately 1.5 m away from the particle, where the intensity increased to above the original intensity. This may be due to a short, free filament landing above the filament. In the second case, th e intensity recovery followed the same trend of intensity along the length as the original filament, reaching appr oximately 50% to 75% of the initial intensity. In both cases, after three minutes of recovery, the fluorescent intensity decreased slightly.

PAGE 118

118 Photobleaching is most likely occurring again afte r this time period, despite the short exposure time, causing the fluorescent intensity to decrease. The fluorescent intensity was also measured as a function of time. We fitted the fluorescent intensity as a function of time and fo und both the equilibrium intensity and the rate constant at various points along the filament length. In Figures 5-6A a nd B, corresponding to the filament represented in Figure 5-5A, neither th e rate constant nor the equilibrium intensity followed a clear pattern with respect to the length of the filament. The first two rate constants may be lower because the points were located near the center of the bead, which did not photobleach completely. In Figures 5-6C and D, which correspond to the filament in Figure 5-5B, the rate constant decreased exponentially over the length of the filament, reaching a final value assumed to correspond to the background rate of recovery. This implies that recovery near the bead is faster, i.e., monomers are incorporating in the filament at the beads surface first. Based on the initial fluorescent intensity, the region ne ar the bead seems to have a high concentration of monomers, which could be locally available to the (+)-ends of polymerizing filaments. The equilibrium intensity remained near the same ratio (compare d to the initial intensity before photobleaching) along the length of the filament, except fo r a decrease between 0.5 and 1 m. 5.3.2 Single Actin Filaments From these results, it is difficult to determine clearly if filaments are incorporating at the surface of the particle through fila ment end-tracking motors. Ther efore, the brain extract buffer was diluted to reduce the number of filame nts per bead and samples were fixed with gluteraldehyde to observe a sing le filament attached to a bead (Figure 5-7). These samples showed single filaments that were present on particles bound to the surface. The filaments were

PAGE 119

119 all swept in the direction of flow during the fixi ng step, potentially causing two filaments (on the same bead) to appear as one. We next attempted to bind filaments with att ached beads to the surf ace to observe particle motility. NEM-myosin surfaces bound many filame nts, but beads were hard to image from solution because they were small with large ther mal motion (Figure 5-8). An enlarged image of the modified surfaces shows filame nts with a corresponding bead, but it is unclear if they were attached or simply near each other by chance. The images from this experiment are difficult to interpret because many filaments may be free fila ments in solution that bound to the surface but are not associated with a bead. Motility assays with and without beads appeared very similar in TIRF when looking at the fluorescently-labeled F-actin, confirming that F-actin was present in large concentrations in the extr act (Figure 5-9). The actin mono mer concentration in the solution is high (> 5 M), which provided ideal conditions for the formation of free filaments. 5.4 Discussion We have attempted to propel small partic les by single filament elongation using the ActA/VASP complex. We found th at the fluorescent intensity al ong the length of the filaments decreased exponentially from the center of the particle to the end of the filaments, inferring that the polymerization may be occurring at the bead surface. Fluorescent recovery after photobleaching showed that, in the case of one filament, faster r ecovery rates o ccurred near the surface of the bead. Other filaments either did not recover or had different intensity trends compared to the initial filament, leaving th e photobleaching results inconclusive. If the elongation of all filaments was occurring by inserti onal polymerization, we would expect that the recovery rate would continue to decrease along the filament length because monomers can only add to the (+)-end of the filament.

PAGE 120

120 Formation of the actin asters potentially have multiple mechanisms at work that are difficult to distinguish from one another through fluorescent microscopy. Single actin filaments are only 7 nm in diameter, making it possible that the free filaments in solution and the background fluorescence of the surface in TIRF may be distorting some of the observed trends. Therefore, insertional polymerization may not be solely responsible for the recovery of fluorescence in our photobleaching experiments. Another explanation of the photobleaching results could be that multiple filaments are li ned up with each other, appearing as one, more intense filament in certain re gions. New filaments may be dropping to the surface as seen in Figure 5-3 and randomly landing on or around fi laments attached to the beads. In addition, two previously reported studies add to the possible explanations of the formation of actin stars and growth on beads. Brown, et al. observed actin stars on polylysine-coated 1-m diameter polystyrene beads, which can nucleate polar assembly of filaments. In this case, through myosin subframent S1 labeling and observation with an electron microscope, the filaments were shown to be attach ed to the bead at the (-)-end and therefore, polymerized outward by adding monomers to the fila ment (+)-end (118). Ou r results hint that polymerization is occurring at the bead, but we ha ve not conclusively eliminated the possibility of filaments attached to the particles at their (-)-end by some other mechanism. Vignjevic, et al. observed star-like actin structures with bundled filaments, not single-filaments, creating the arms. These stars demonstrated organizati on of both lamellapodia and filopodia and were dependent on the concentrations of Arp2/3 and capping protein (119). Through fluorescent microscopy, we can not distingu ish between single filaments and bundles of a few filaments. Bundling proteins are likely present in the cell extracts, making it possible for bundles of filaments to form.

PAGE 121

121 Diluting the extract seem ed to consistently produce beads w ith single or a few filaments. Insight into the mechanism has recently been ga ined with the use of electron microscopy (EM) images and TIRF color-change experiments (Sturm C., J. Phillips, W. Zeile, K. Interliggi, R. Dickinson, and D. Purich, unpublished). Details of filaments, such as the difference between bundles and single filaments are ha rd to resolve in TIRF, ther efore, EM was used. Under specific conditions, a majority of particles attached to filame nts only had one filament per particle. Furthermore, by exchanging the actin from a green fluorescent la bel to a red fluorescent label (or vice-versa) in a flow-cell, the colo r of the labeled actin added second was always closest to the bead, indicating insertional polym erization (i.e. the (+)-e nd is attached to the surface of the bead). In most cases, the filaments grew to an average length of 500 nm for 10 seconds, corresponding to an elongation rate of approximate ly 3 m/min (Sturm, C., J. Phillips, W. Zeile, K. Interliggi, R. Dickinson, and D. Purich, unpublished). The length may be controlled by the presence of capping protein in the extract and could possibly be optimized through varying the concentr ation of capping protein. For the purpose of control over this system, it may be necessary to use pure component systems which include actin, Arp2/3, ADF, capping protein, VASP, profilin, and -actinin (115). A pure component system would provide control over protein co ncentrations, and coul d help to polymerize filaments for longer timeand length-scales, essentially broadening their application in nanodevices. When NEM-myosin surfaces were used, visualization was difficult because many actin filaments did not correspond to a bead. Due to th e extent of nonspecific actin polymerization, it could not be confirmed that actin filament tails were attached to 50-nm diameter beads when applied to the NEM-myosin surface. Many filame nts are most likely spontaneously forming due

PAGE 122

122 to the high concentration of actin, as indicated by the control sample in which no beads were added. Solutions to preventing nonspecific fila ment formation include the use of profilin or capping proteins. Insight into the mechanism of actin-based motility suggests that we are making strides toward single filament elongation that can propel small particle s end-tracking motors, such as formins and possibly ActA/VASP. Single filaments have the adva ntage over large rocket tails because they are more flexible, making it easier to manipulate their paths on patterned surfaces. Motile particles would need to be small enough so they do not effect th e fluctuations of the attached, elongating filament end. The limitation of these experiments is visualization of both the 50-nm diameter particle and the actin filament in fluorescence at the same time. Biochemical optimization of the system could lead to be tter visualization and the development of single-filament actuators for use in bionanodevices.

PAGE 123

123 Figure 5-1. Growth of actin fi laments/bundles on 50-nm diameter beads. Initially, the 50-nm beads bound to the surface become surrounded by an actin network. The number of filaments and, to some extent, the length of the filaments becomes larger and longer with time. Experiments used full extract st rength with an addition of 5 M of Oregon green-labeled actin (scale bar = 5 m).

PAGE 124

124 Figure 5-2. Fluorescent intens ity of actin filaments/bundles. A) Enlarged image from Figure 5-1 of 50-nm bead bound to the surface. The fluorescent intensity is greatest in the center of the bead (scale bar = 3 m). B) Fluorescent intensity as a function of length along the filament. The numbers in the legend correspond to the filament labels in A. A line-scan across the filaments show that for all four cases, the intensity decreased for approximately 3 m of length, where it ultimately levels out to the background intensity. Line-scans began at the beginning of each pr otrusion at the apparent surface of the bead.

PAGE 125

125 Figure 5-3. Actin asters recove ry after photobleaching. A) Initi al image of asters of actin on 50-nm diameter beads. B) After photobleaching, only a small fluorescent signal is present at the center of th e bead. C) Over time, fl uorescence is regained and filaments reappeared on the 50-nm beads. Fi laments not associated with a bead also reappeared on the surface. Images were taken 1.5 minutes apart (scale bars = 5 m).

PAGE 126

126 Figure 5-4. Enlarged image of asters reappear ing after photobleaching. A) Initial image of asters of actin on 50-nm beads (from Figur e 5-3A). B) After photobleaching, only a small fluorescent signal is present at the center of the bead. C) Over time, fluorescence is regained and filaments reappeared on the 50-nm beads. The filaments that appear during the rec overy of photobleaching may co rrespond to initial filaments seen in A. Images were taken 1.5 minutes apart (scale bars = 2 m).

PAGE 127

127 Figure 5-5. Fluorescent intensity of photobleached filaments. A) Fluorescent intensity of filament 1 (Figure 5-4A) as a function of tim e. At length = 0 m to 1.5 m, recovery with respect to length followed the initial trend. At length greater than 1.5 m, fluorescent intensity exceeded the initial in tensity and also increased slightly with increasing length. B) Fluorescent intensity of filament 2 (Figur e 5-4B) as a function of time. The intensity of the filament as a function of length followed the initial intensity, before photobleaching.

PAGE 128

128 Figure 5-6. Recovery rates and equilibriu m intensities of photobleached filaments. A) Fluorescent recovery rate of filament 1 (Figure 5-4A) as a function of position of the filament from the center of the bead. B) The ratio of the calculated equilibrium intensity and the initial intensity before photobleaching of filament 1 (Figure 5-4A). Note that the intensity recovered betw een 1.5 and 2 m is greater than 1, corresponding to the region of the filament that was brighter than the initial filament. C) Fluorescent recovery rate of filament 2 (Figure 5-4B). The rates follow an exponential trend as the length away from th e bead increases. D) The ratio of the calculated equilibrium intensity and the initial intensity of filament 2 (Figure 5-4B). With the exception of intensities between 0.75 and 1 m, the equilibrium intensities to initial intensity ratio seem to be within 0.35 and 0.55.

PAGE 129

129 Figure 5-6. Continued

PAGE 130

130 Figure 5-7. 50-nm diameter beads bound to surf ace with single filaments or bundles attached. Experiments were fixed with gluteraldehyde Optimization of conditions with the extract (diluted 4:3 in brain extract buffer) led to a reduced number of filaments per bead (scale bar = 5 m).

PAGE 131

131 Figure 5-8. 50-nm diameter beads in soluti on on NEM-myosin surfaces. A) Instantaneous image of fluorescent 50-nm diameter partic les, which are constantly moving in and out of the focal plane with time sin ce they are not bound to the surface. B) Rhodamine-labeled actin filament s bound to NEM-myosin surface in same field-of-view. C) Overlay of images A and B. The large number of filaments make it difficult to see any corresponding beads a nd filaments (scale bar = 10 m). D) Enlarged image of beads near filament ends. It is unclear if the beads are attached to the filaments or if they are near each other (scale bar = 3 m).

PAGE 132

132 Figure 5-9. Actin motility assay with and with out beads on NEM-myosin surfaces. A) Sample containing no beads still produced fila ments in solution which bind to the NEM-myosin treated surface. B) Sample containing beads produced and bound approximately the same amount of filaments as a sample with no beads. Both samples have 5 M of Oregon green-la beled actin (scale bar = 5 m).

PAGE 133

133 CHAPTER 6 SUMMARY AND FUTURE WORK Applications that involve the exploitation of biomolecu lar m otors are continuously expanding to include a broad spectrum from bi osensors for single molecule detection to production of nanoelectromechanical devices. Several methods have already shown promise towards these types of applications, including the gliding of actin filaments or microtubules on myosinor kinesin-modified surf aces, respectively, and the move ment of cargo-carrying myosin or kinesin on immobilized filaments (32). These techniques have yet to explore the potential of actin polymerization and actin-based motility as possibilities for nanoscale transport. 6.1 Single Actin Filaments The work presented in Chapter 2 demonstrates a technique that aligns polymerizing actin filaments on tracks with the poten tial for transport of material through cargo-carrying myosin or end-tracking motors bound to filament (+)-ends. Single filaments can potentially attach to end-tracking motors bound to cargo to propel sma ll nanoparticles in an aligned fashion for suitable applications that do not change the therma l fluctuations of the filament. Such particles may include 50-nm diameter particles, antibodies, or quantum dots (2, 4, 14). One important aspect of this assay is that the density of f ilaments on the surface can be easily controlled. We demonstrated that the filament density decreased on surfaces with the additi on of profilin to the actin solution. Higher filament density can be achieved by increasing actin concentration and actin nuclei, potentially with the addition Arp2/3. Our experiments showed that elongating act in filaments bound to NEM-myosin tracks aligned with the track edge in a manner depe ndent on the track width and NEM-myosin surface density. We concluded that the alignment of filaments at track boundaries was facilitated by filament thermal fluctuations, based on the following results. Alignment of actin filaments was

PAGE 134

134 inversely proportional to the track width, with the trend leveling off to slightly above random alignment at 10-m wide tracks. This can be ex plained by the higher ratio of track edges to track area for the narrower track widths. This in crease in ratio causes higher alignment of bound filaments through more frequent in teractions with the track edge Filaments near track edges also demonstrated a bias in alignment, regardle ss of the track width. This is supported by the conclusion that the probability of an actin fila ment rebinding once crossi ng the track edge was dependent on the angle with which the filame nt crossed the edge. No filaments elongating passed the edge at angles great er than 60 realigned. These results indicate that microcontact printing NEM-myosin is a viable method for the creation of filament-binding tracks on glass su rfaces. One downfall included the diffusion of NEM-myosin molecules from the tracks to the passivated regions, which most likely occurred because the NEM-myosin is adsorbed to the surf ace, not covalently attached (82). Although this did not seem to affect the fi nal results, some weak binding (to one or a few NEM-myosin molecules only) occurred in the BSA regions of the surface. Another problem was that deformations of the stamp occurred, creating tr acks of varying sizes when stamped with the PDMS pattern, as well as some track s without center regions (120). The proposed mechanism for the alignment of filaments was supported through simulations, which used the bending energy of the filament to determine filament shapes and behavior (108). The simulations predicted the sa me effects of track width, myosin concentration and rebinding probability on alignmen t of filaments as were found experimentally in Chapter 2. Experimental variations that may account for di fferences between simulations and experiments include variations in surface density and filament -binding activity of the substratum. We also looked at the effect of persis tence length on the filaments, fi nding that the persistence length

PAGE 135

135 does play an important role in the rebinding of the filaments. Because our proposed mechanism requires bending of filaments, the persistence le ngth was expected to play a key role in the filament alignment. The simulations will allow for the future exploration of the effect of various conditions, including differe nt shaped tracks (21). 6.2 Actin-Based Motility We have reproduced previous methods that use the surface protein, ActA, to induce actin-based motility by forming ac tin rocket tails that propel Listeria and particles in vitro. NEM-myosin was used to confine actin rocket ta ils to glass surfaces and subsequently, contain the path of the motile object near the surface wit hout affecting the velocity. The binding of the actin rocket tails to the surface may be a result of both the surface and the structural features of the rocket tail. Specifically, the rocket tails contain single actin filaments (or small bundles) protruding from the sides of the tail, presum ably produced by Arp2/3. The NEM-myosin molecules may bind to these filaments in addition to the exterior filaments in the rocket tail bundle. The ability of the surface to contain these larg e structures and reduce fluctuations in their paths is a significant step toward the use of the ActA/VASP complex and end-tracking motors in bionanodevices. Elucidating the mechanism by whic h binding occurs may help to optimize the system for applications. We attempted to guide particle propulsion fu rther with the use of channels on glass surfaces. The channel design requi res features such as width, material used and height to sufficiently prevent the actin rock et tail and attached object from escaping, an important property for transporting particles in devices. Further optimization of the channel design is needed to determine the effectiveness of this system for applications. We expect that this system can be applied to small particles with single filaments attached. Single filament propulsion is difficult to achie ve but extremely useful for bionanodevices. We

PAGE 136

136 have successfully attached ActA-coated beads to surfaces and observed single actin filament growth from these beads, alt hough the work presented here does not conclusively confirm the mechanism by which the filaments are attached. We have shown the difficulties in visualizing and confining single actin filaments to beads in a cell extract on NE M-myosin surfaces. Difficulties include a high density of filament s (not produced by ActA/VASP) and imaging a fluctuating bead, both inhibiting th e ability to definitively observ e a motile bead attached to an elongating single filament. 6.3 Recommendation for Future Work 6.3.1 Filament-Binding Tracks The effect of microcontact-printed tracks on the alignment of elongating actin filaments has been characterized in the presented work Further work should be aimed towards the optimization of the mircrocontact printing of NEM-myosin. The technique of patterning proteins is complicated in that the activity of the prot ein, which may be depende nt on the structure and position of the protein, must be conserved. Optimization of microcontact printing may provide a way to ensure the position of NEM-myosin is at its most effective, therefore, increasing the binding activity and maintaining the integrity of the pattern (20). Modifications that could be e xplored include covalently binding NEM-myosin to an APES-treated surface stamped with gluteraldehyde, ensuring that the myosin heads are exposed. In addition, it may be beneficial to work with heavy-meromyosin, a smaller, still active part of the myosin molecule. The smaller molecule may make it easier to maintain activity and structural integrity, especially if the myosin is covalently bound to a surface (18). Another possibility is to exploit biotin-streptavidi n interactions, which are commonly used in microcontact printing and easily attached to protei ns (18, 24). These techniques may reduce the diffusion of myosin from the tracks to the pa ssivated regions while making the protein more

PAGE 137

137 effective. Stamp deformation, another problem associated with microcontact printing, may be reduced by optimizing and standardiz ing the pressure applied to th e stamp during the transfer of the proteins. Other work should be focused towards varying the shape of the filament-binding regions to include curves and corners to determine the effect on the alignment of filaments. In addition, controlling density of th e filaments may help to optimize alignment and elongation. We have already determined that profilin lowers the density on the surface. Other proteins that may have an effect on the density or actin network include capping protein, whic h increases the (+)-end critical concentration to maintain filament lengths, Arp2/3, which increases nucleation and also produces branching for networking, and -actinin, which cross-links filaments into a continuous network (39). All of these proteins provide advantages to immobilized filament assays by controlling the characteristics of the filament s on the surface, including the potential for lengthening the path for myos in-based transport. 6.3.2 Three-Dimensional Surfaces for Larger Structures Further investigation into the effect of microcontact printing on actin rocket tails propelling Listeria and larger beads in vitro is recommended. Howeve r, it is unlikely that microcontact printing alone will help to align actin rocket tails, which consist of thousands of filaments. Three-dimensional devices in comb ination with NEM-myosin surfaces are expected to be more apt to guide particle propulsion. These types of devices, with modified surfaces and topographical features, have been most successf ul at the guidance of sliding microtubules compared to systems using only one of the tw o features (16, 105). Further work on the optimization of these devices woul d likely provide the means to gui de elongation of actin rocket tails and their attached particles. The height of the topography, as indicated by our experiments, is an important parameter to be optimized so that the particles do not climb over the walls, but

PAGE 138

138 rather turn at the walls, following the designated path. Undercuts on the top of the channel walls may further ensure that beads remain near the surface (17). In addition, devices may need less exposed glass than those shown in Chapter 4, creating longer channels that span the entire flow-cell. This design should in crease the chances that tails bi nd to surfaces inside channels. 6.3.2 Use of End-Tracking Motors To obtain more control over the bioche mistry involved in the ActA/VASP and end-tracking motor systems, it is necessary to test pure component systems, which have previously been reported to sustain actin-based mo tility (115). This is particularly important for the single-filament processive motors, which prod uced more filaments than just those from the ActA-coated beads (due to a high actin concen tration). Observation of single-filament elongation is important not only fo r biodevices, but also for the elucidation of the mechanism by which ActA/VASP produces force. Although this mechanism is still under debate, it remains that the force generated can be advantageous for in vitro applications. The Brownian Ratchet model allows for the release of energy of monomer addition (2 kT) (86). The actoclampin mechanism allows for the energy released from ATP hydrolysis to also be used, which would provide 16 kT of accessible energy and a corresponding opposing force of 12 pN (26). Since formins produce a single, unbranched filame nt, they provide a simpler system for the use of end-tracking motors, and should be consider ed for applications in bionanotechnology. In previous reports, the stall force and buckling force for single actin filaments were measured by tethering the filament (+)-ends to formins and attaching another point of the filament to the surface with NEM-myosin. The buckling force of the filaments is proportional to its stiffness and inversely proportional to its length. Therefore, the shor test filament formed (0.75 m), produced a force of 1.3 pN (94). The forces from formins, ActA/VA SP and other end-tracking motors can provide many advantageous to microand nano-scale applications. In vivo, motors

PAGE 139

139 are efficient and essential to many cellular pr ocesses, making them optimal for engineering future bionanodevices.

PAGE 140

140 APPENDIX MATLAB CODE This progra m simulates the el ongation of actin filaments on patterned, filament-binding surfaces. This program is designed to run mu ltiple filaments in a loop, with one filament elongating per iteration. We first looked at the effect of the filament probability of rebinding when leaving the surface by setting the initial pos ition to 4.99 m with the track edge located at 5 m. We then varied the number of modes, the binding probability constant, the persistence length, the final total length of the filaments, and the step si ze, ds. We also looked at the alignment of filaments as a function of track widt h, where the initial position and initial angle of the filaments were randomly selected. We varied the binding probability c onstant, the number of modes, and the persistence length. Lengths and parameters were estimated and varied based on experimental values. %This program simulates the elongation of filaments on patterned, %filament-binding surfaces clear all rand('seed' sum(clock)); %Seeds random number generators in MATLAB randn( 'seed' sum(clock)); %Number of Runs ns = 20; %Number of filaments per run nt = 15*100 %Maximum length per filament Number of steps per filament nsnt = ns*nt; %Number of maximum possible iterations %Set Initial Matrices x=zeros(1,nsnt); %Initial x, y matrices that determine final filament shape y=x; xcross=zeros(1,nsnt); %Initial x, y matrices that determine ycross=x; %Instantaneous position of elongating filament end xcross2 = x; %Second to last instantaneous position of filament end ycross2 = y; xb=zeros(1,nsnt); %Initial x, y matrices at which binding occurs yb=xb; thend = zeros(1,nsnt); %Initial theta matrix at which binding occurs ibin=xb; %Iteration at which binding occurs %mostly for troubleshooting purposes Lbind = xb; %Length of filament binding

PAGE 141

141 ibind=1 %Set initial iteration at which binding occurs k = 1; %Determine Length of Filament 1 fillen = (15*rand(1,1))+1 %Select random length less than %15 microns of first filament nx=round(fillen*100) %Number of steps for initial filament %Repeat Loop for Each Filament for count = 1:ns %For filament 1 through filament ns dt=1; %Time increment (seconds) ds=.01; %Change in length (microns) %Generate random number matrices for filament count rnd = rand(1,nx); % Rnd(x) is used to test the probability of binding rndn=randn(1,nx); %Input Variables nmod = 2 % Number of modes kp=0.01 % Binding probability scaled to kb^-1 (microns^-1*seconds^-1) width = 8; % Input track width space = 20; % Input space between tracks lam = 10; % Persistence length (microns) v=0.01; %Elongation rate of filaments (microns/sec) %Randomize initial starting point and theta, initial binding point x(k) = 0; %Initial x position is 0 y(k)= (width-.01)*rand-(width/2); % Initial y position is randomly chosen yb(k)=y(k); %Binding occurs in the initial position xb(k)= x(k); %xb = x(0) and yb = y(0) thend(k) = (((90*pi/180))*rand); % Direction of filament at time = 0 L = 0; % Length of unbound region initially xcross(k) = 0; %Binding occurs in the initial position ycross(k)= y(k); %xcross(0) = x(0) and ycross(0) = y(0) xcross2(k) = 0; ycross2(k)= y(k); initial_position = y(k); %Print initial position in command window initial_angle = thend(k); %Print initial angle in command window %Loop Increases Free Filament Length for i=k:nx %k = initial iteration of filament %Filament 1 starts at k =1, nx = filament length rnds=randn(1,nmod); %Random variable selected for each mode L=L+v*dt; %Length is increased by ds = v dt %Determine if Binding Occurs if rnd(i) < kp*dt*L %Check if binding can occur based on %Filament length and probability constant %Equal probibility along length Lbin = rand(1,1)*L; %Determine random position on %unbound length that binding occurs if Lbin > ds %Make sure that Lbin is larger than ds %program can not continue (matrix can not be formed)

PAGE 142

142 s=0:ds:L; %Create matrix of all positions, s, along free filament sbind = 0:ds:Lbin; %Create matrix of all postions, sbind, %along portion of filament that is about to bind %Calculate maximum amplitudes for each mode sign = sqrt (2*L./(((0:(nmod-1))+0.5).^2)/lam/pi^2); modes = sign.*rnds; %Calculate actual amplitudes using %random numbers less than 1 theta=0*s; %Create initial matrix theta(s) for im=1:nmod %Calculate theta for every point, s theta = theta + modes(im)*sin((im-1+.5)*pi*s/L); %theta(1) = 0 %Sum all modes end %Determine x and y points of filament from theta and s xp=zeros(1, length(s)-1); %Set initial matrices for xp and yp yp=xp; for j=2:length(s) %j starting at 2 eliminates the point 0,0 from xp %Trapezoid rule used to integrate xp(j-1)=(ds/2)*sum((cos(theta(1:(j-1))))+(cos(theta(2:j)))); yp(j-1)=(ds/2)*sum((sin(theta(1:(j-1))))+(sin(theta(2:j)))); end %Determine postions with rotation of axis rotated %with respect to the initial theta (binding position of filament) %x(1) and y(1) are already set to 0 x(ibind+1:ibind+(length(xp)))=x(ibind)+xp*cos(thend(i))yp*sin(thend(i)); y(ibind+1:ibind+(length(xp)))=y(ibind)+xp*sin(thend(i))+yp*cos(thend(i)); %Check if binding (Lbind) occurs within track %Create wave function to allow for the case of multiple tracks if cos((y(ibind+length(sbind)-1)/((width+space)/2))*pi)cos(((width)/(width+space))*pi)>0 %Record filament end positions ycross(i)=y(ibind + length(xp)); xcross(i)= x(ibind + length(xp)); ycross2(i)=y(ibind-1 + length(xp)); xcross2(i)= x(ibind-1 + length(xp)); ibind=ibind+length(sbind)-1 %Increase ibind (iteration number %that binding occurs)to new binding position ibin(i)=ibind; %Create matrix of iterations for trouble-shooting xb(i)=x(ibind); %Place x, y position of binding in xb and yb yb(i)=y(ibind); Lbind(i)= Lbin; %Record the length of filament that binds %Calculate angle of the filament segment at position of binding thbind(i)= thend(i)+ theta(length(sbind)-1); L= L-Lbin %Update length of unbound region thend(i+1) = thbind(i); %Update angle at which binding occurs %Free filament will now undulate around

PAGE 143

143 else %If filament binding is NOT within track, no binding occurs ibind=ibind %Ibind remains the same if no binding occurs thend(i+1) = thend(i); %Thend remains the same %(binding angle of last binding spot) %Record filament end positions ycross(i)=y(ibind + length(xp)); xcross(i)= x(ibind + length(xp)); ycross2(i)=y(ibind-1 + length(xp)); xcross2(i)= x(ibind-1 + length(xp)); end else %If Lbind < ds (not enough binding length) %Repeat calculation of filament shape (Lines 80, 83-97) s=0:ds:L; sign = sqrt (2*L./(((0:(nmod-1))+0.5).^2)/lam/pi^2); modes = sign.*rnds; theta=0*s; for im=1:nmod theta = theta + modes(im)*sin((im-1+.5)*pi*s/L); end %Determine x and y points of filament from theta and s xp=zeros(1,length(s)-1); yp=xp; for j=2:length(s) xp(j-1)=(ds/2)*sum((cos(theta(1:(j-1))))+(cos(theta(2:j)))); yp(j-1)=(ds/2)*sum((sin(theta(1:(j-1))))+(sin(theta(2:j)))); end % Calculate positions with rotation x(ibind+1:ibind+(length(xp)))=x(ibind)+xp*cos(thend(i))yp*sin(thend(i)); y(ibind+1:ibind+(length(xp)))=y(ibind)+xp*sin(thend(i))+yp*cos(thend(i)); thend(i+1) = thend(i); %Thend remains the same %Record filament end positions ycross(i)=y(ibind + length(xp)); xcross(i)= x(ibind + length(xp)); ycross2(i)=y(ibind-1 + length(xp)); xcross2(i)= x(ibind-1 + length(xp)); end else %If binding probability is less than the random number generator %Repeat calculation of filament shape (Lines 80, 83-97) s=0:ds:L; sign = sqrt (2*L./(((0:(nmod-1))+0.5).^2)/lam/pi^2); modes = sign.*rnds; theta=0*s; for im=1:nmod theta = theta + modes(im)*sin((im-1+.5)*pi*s/L); end %Determine x and y points of filament from theta and s xp=zeros(1,length(s)-1); yp=xp; for j=2:length(s) xp(j-1)=(ds/2)*sum((cos(theta(1:(j-1))))+(cos(theta(2:j)))); yp(j-1)=(ds/2)*sum((sin(theta(1:(j-1))))+(sin(theta(2:j))));

PAGE 144

144 end % Calculate positions with rotation x(ibind+1:ibind+(length(xp)))=x(ibind)+xp*cos(thend(i))yp*sin(thend(i)); y(ibind+1:ibind+(length(xp)))=y(ibind)+xp*sin(thend(i))+yp*cos(thend(i)); thend(i+1) = thend(i); %Thend remains the same %Record filament end positions ycross(i)=y(ibind + length(xp)); xcross(i)= x(ibind + length(xp)); ycross2(i)=y(ibind-1 + length(xp)); xcross2(i)= x(ibind-1 + length(xp)); end k = k+1; %Increase k (iteration number for entire simulation end %End elongation of one filament ibind = k; %New filament binds at the first iteration of k fillen = (15*rand(1,1))+1 %Set length of new filament nx=round(fillen*100) % Set number of iterations for new filament nx = k + nx; % Increase total number of iterations end xprint = x(1:10:nsnt); %Create matrix of final x and y coordinates and theta yprint = y(1:10:nsnt); thendi = thend(1:nsnt); %Create Excel spreadsheets matrixcross = [thendi', xcross', ycross', xcross2', ycross2',ibin', thbind', xb', yb', Lbind' ]; matrix2 = [xprint', yprint']; xlswrite( '8Oct1_C' matrix2, '6'); %Send final positions of filament xlswrite( '8Oct1_PC' matrixcross, '6'); %Send instantaneous positions of %filaments and binding information

PAGE 145

145 LIST OF REFERENCES 1. Howard, J. 2001. Mechanics of motor proteins and the cytoskeleton. Sinauer, Sunderland, MA. 2. Bachand, G. D., S. B. Rivera, A. K. Boal J. Gaudioso, J. Liu, and B. C. Bunker. 2004. Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins. Nano Lett. 4:817-821. 3. Bohm, K. J., R. Stracke, P. Muhlig, and E. Unger. 2001. Motor protein-driven unidirectional transport of mi crometer-sized cargoes acros s isopolar microtubule arrays. Nanotechnology. 12:238-244. 4. Mansson, A., M. Sundberg, M. Balaz, R. Bunk, I. A. Nicholls, P. Omling, S. Tagerud, and L. Montelius. 2004. In vitro sliding of actin filaments labe lled with single quantum dots. Biochem. Biophys. Res. Commun. 314:529-534. 5. Muthukrishnan, G., B. M. Hutchins, M. E. Williams, and W. O. Hancock. 2006. Transport of semiconductor nanocrystals by kinesin molecular motors. Small. 2:626-630. 6. Suda, H., and A. Ishikawa. 1997. Accelerativ e sliding of myosin-c oated glass-beads under suspended condition from actin paracrystal. Biochem. Biophys. Res. Commun. 237:427-431. 7. Suzuki, N., H. Miyata, S. Ishiwata, and K. Kinosita, Jr. 1996. Preparation of bead-tailed actin filaments: Estimation of the tor que produced by the sliding force in an in vitro motility assay. Biophys. J. 70:401-408. 8. Bunk, R., J. Klinth, L. Montelius, I. A. Ni cholls, P. Omling, S. Tagerud, and A. Mansson. 2003. Actomyosin motility on nanostructured surfaces. Biochem. Biophys. Res. Commun. 301:783-788. 9. Hess, H. 2006. Materials science. Toward devices powered by biomolecular motors. Science. 312:860-861. 10. Hess, H., G. D. Bachand, and V. Vogel. 2004. Powering nanodevices with biomolecular motors. Chemistry. 10:2110-2116. 11. Hess, H., J. Clemmens, C. Brunner, R. Doot S. Luna, K. H. Ernst, and V. Vogel. 2005. Molecular self-assembly of "nanowires"a nd "nanospools" using active transport. Nano Lett. 5:629-633. 12. Jia, L., S. G. Moorjani, T. N. Jackson, a nd W. O. Hancock. 2004. Mi croscale transport and sorting by kinesin molecular motors. Biomed. Microdevices. 6:67-74. 13. Lin, C. T., M. T. Kao, K. Kurabayash i, and E. Meyhofer. 2006. Efficient designs for powering microscale devices with nanoscale biomolecular motors. Small. 2:281-287.

PAGE 146

146 14. Ramachandran, S., K. H. Ernst, G. D. Bachand, V. Vogel, and H. Hess. 2006. Selective loading of kinesin-powered molecular shuttles with protein cargo a nd its application to biosensing. Small. 2:330-334. 15. Sundberg, M., R. Bunk, N. Albet-Torres, A. Kvennefors, F. Persson, L. Montelius, I. A. Nicholls, S. Ghatnekar-Nilsson, P. Omling, S. Tagerud, and A. Mansson. 2006. Actin filament guidance on a chip: Toward hi gh-throughput assays and lab-on-a-chip applications. Langmuir. 22:7286-7295. 16. Clemmens, J., H. Hess, R. Doot, C. M. Matzke, G. D. Bachand, and V. Vogel. 2004. Motor-protein "Roundabouts": Microtubules moving on kinesi n-coated tracks through engineered networks. Lab Chip. 4:83-86. 17. Hess, H., C. M. Matzke, R. K. Doot, J. Clemmens, G. D. Bachand, B. C. Bunker, and V. Vogel. 2003. Molecular shuttles operating unde rcover: A new photolit hographic approach for the fabrication of structured surfaces supporting directed motility. Nano Lett. 3:1651-1655. 18. Manandhar, P., L. Huang, J. R. Grubich, J. W. Hutchinson, P. B. Chase, and S. Hong. 2005. Highly selective directed assembly of functional actomyosin on Au surfaces. Langmuir. 21:3213-3216. 19. Nicolau, D. V., H. Suzuki, S. Mashiko, T. Taguchi, and S. Yoshikawa. 1999. Actin motion on microlithographically functionali zed myosin surfaces and tracks. Biophys. J. 77:1126-1134. 20. Sundberg, M., M. Balaz, R. Bunk, J. P. Rose ngren-Holmberg, L. Montelius, I. A. Nicholls, P. Omling, S. Tagerud, and A. Mansson. 2006. Selective spatial loca lization of actomyosin motor function by chemical surface patterning. Langmuir. 22:7302-7312. 21. Suzuki, H., A. Yamada, K. Oiwa, H. Nakayama, and S. Ma shiko. 1997. Control of actin moving trajectory by patterned pol y(methylmethacrylate) tracks. Biophys. J. 72:1997-2001. 22. Reuther, C., L. Hajdo, R. Tucker, A. A. Kasprzak, and S. Diez. 2006. Biotemplated nanopatterning of planar surf aces with molecular motors. Nano Lett. 6:2177-2183. 23. Asokan, S. B., L. Jawerth, R. L. Carroll, R. E. Cheney, S. Washburn, and R. Superfine. 2003. Two-dimensional manipulation and orie ntation of actin-myosin systems with dielectrophoresis. Nano Lett. 3:431-437. 24. Huang, L., P. Manandhar, K. E. Byun, P. B. Chase, and S. Hong. 2006. Selective assembly and alignment of actin filaments with desired polarity on solid substrates. Langmuir. 22:8635-8638. 25. Dickinson, R. B., L. Caro, and D. L. Purich. 2004. Force generation by cytoskeletal filament end-tracking proteins. Biophys. J. 87:2838-2854.

PAGE 147

147 26. Dickinson, R. B., and D. L. Purich 2002. Clamped-filament elongation model for actin-based motors. Biophys. J. 82:605-617. 27. Romero, S., C. Le Clainche, D. Didry, C. Egile, D. Pantaloni, and M. F. Carlier. 2004. Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis. Cell. 119:419-429. 28. Dabiri, G. A., J. M. Sanger, D. A. Po rtnoy, and F. S. Southwick. 1990. Listeria monocytogenes moves rapidly through the hos t-cell cytoplasm by inducing directional actin assembly. Proc. Natl. Acad. Sci. U.S.A. 87:6068-6072. 29. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109:1597-1608. 30. Tirnauer, J. S., J. C. Canman, E. D. Salm on, and T. J. Mitchison. 2002. Eb1 targets to kinetochores with attached, polymerizing microtubules. Mol. Biol. Cell .13:4308-4316. 31. Hess, H., and V. Vogel. 2001. Molecular shuttles made from motor proteins: Active transport in non-biolog ical environments. Reviews in Molecular Biotechnology. 82:67-85. 32. Bakewell, D. J. G., and D. V. Nicolau. 2007. Protein linear molecular motor-powered nanodevices. Aust. J. Chem. 60:314-332. 33. Hess, H., J. Clemmens, J. Howard, and V. Vogel. 2002. Surface imaging by self-propelled nanoscale probes. Nano Lett. 2:113-116. 34. Laki, K., W. J. Bowen, and A. Clark. 1950. The polymerization of proteins; adenosine triphosphate and the pol ymerization of actin. J. Gen. Physiol. 33:437-443. 35. Mommaerts, W. F. 1951. Reversible polymer ization and ultracentrifugal purification of actin. J. Biol. Chem. 188:559-565. 36. Otterbein, L. R., P. Graceffa, and R. Dominguez. 2001. The crystal structure of uncomplexed actin in the ADP state. Science. 293:708-711. 37. Straub, F. B., and G. Feurer. 1950. Adenos inetriphosphate the f unctional group of actin. Biochim. Biophys. Acta. 4:455-470. 38. Straub, F. B. 1942. Actin. Stud. Inst. Med. Chem. Univ. Szeged. 2:1-15. 39. Southwick, F. S., and D. L. Purich. 2000. Actin filaments: Self-assembly and regulatory interactions. In Cellular Micr obiology. P. Cossart, P. Boquet, S. Normark, and R. Rappuoli, editors. ASM Press, Washington, D.C. 153-170. 40. Schafer, D. A., M. D. Welch, L. M. Maches ky, P. C. Bridgman, S. M. Meyer, and J. A. Cooper. 1998. Visualization and molecular anal ysis of actin assembly in living cells. J. Cell Biol. 143:1919-1930.

PAGE 148

148 41. Wear, M. A., D. A. Schafer, and J. A. Cooper. 2000. Actin dynamics: Assembly and disassembly of actin networks. Curr. Biol. 10:891-895. 42. Kelleher, J. F., S. J. Atkinson, and T. D. Pollard. 1995. Sequences, structural models, and cellular localization of the actin-related proteins arp2 and arp3 from acanthamoeba. J. Cell Biol. 131:385-397. 43. Alberts, B., A. Johnson, J. Lewis, M. Ra ff, K. Roberts, and P. Walter. 2002. Molecular biology of the cell. Garland Science, New York. 44. Kirschner, M. W. 1980. Implications of tr eadmilling for the stability and polarity of actin and tubulin polymers in vivo. J. Cell Biol. 86:330-334. 45. Neuhaus, J. M., M. Wanger, T. Keiser, and A. Wegner. 1983. Treadmilling of actin. J. Muscle Res. Cell. Motil. 4:507-527. 46. Wegner, A. 1976. Head to tail polymerization of actin. J. Mol. Biol. 108:139-150. 47. Kaufmann, S., J. Kas, W. H. Goldmann, E. Sackmann, and G. Isenberg. 1992. Talin anchors and nucleates actin filaments at li pid membranes. A direct demonstration. FEBS Lett. 314:203-205. 48. Isambert, H., P. Venier, A. C. Maggs, A. Fattoum, R. Kassab, D. Pantaloni, and M. F. Carlier. 1995. Flexibility of actin filaments derived from thermal fluctuations. Effect of bound nucleotide phalloidin and muscle regulatory proteins. J. Biol. Chem. 270:11437-11444. 49. Kas, J., H. Strey, J. X. Tang, D. Finger, R. Ezzell, E. Sackmann, and P. A. Janmey. 1996. F-actin, a model polymer for semiflexible chains in dilute, semidilute, and liquid crystalline solutions. Biophys. J. 70:609-625. 50. Sugimoto, Y., M. Tokunaga, Y. Takezawa, M. Ikebe, and K. Wakabayashi. 1995. Conformational changes of the myosin head s during hydrolysis of ATP as analyzed by x-ray solution scattering. Biophys. J. 68:29S-33S; discussion 33S-34S. 51. Lymn, R. W., and E. W. Ta ylor. 1971. Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry. 10:4617-4624. 52. Rayment, I., H. M. Holden, M. Whittaker, C. B. Yohn, M. Lorenz, K. C. Holmes, and R. A. Milligan. 1993. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 261:58-65. 53. Stossel, T. P. 1993. On the crawling of animal cells. Science. 260:1086-1094. 54. Cassimeris, L., D. Safer, V. T. Nachmias and S. H. Zigmond. 1992. Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes. J. Cell Biol. 119:1261-1270.

PAGE 149

149 55. Southwick, F. S., and C. L. Young. 1990. Th e actin released from profilin-actin complexes is insufficient to account for the increase in f-actin in chemoattractant-stimulated polymorphonuclear leukocytes. J. Cell Biol. 110:1965-1973. 56. Kang, F., D. L. Purich, and F. S. Southwick. 1999. Profilin promotes barbed-end actin filament assembly without loweri ng the critical concentration. J. Biol. Chem. 274:36963-36972. 57. Paavilainen, V. O., E. Bertling, S. Fa lck, and P. Lappalainen. 2004. Regulation of cytoskeletal dynamics by ac tin-monomer-binding proteins. Trends Cell Biol. 14:386-394. 58. Mullins, R. D., J. A. Heuser, and T. D. Pollard. 1998. The interaction of arp2/3 complex with actin: Nucleation, high a ffinity pointed end capping, and formation of branching networks of filaments. Proc. Natl. Acad. Sci. U.S.A. 95:6181-6186. 59. McGough, A. 1998. F-actin-binding proteins. Curr. Opin. Struct. Biol. 8:166-176. 60. Carlier, M. F., V. Laurent, J. Santolini, R. Melki, D. Didry, G. X. Xia, Y. Hong, N. H. Chua, and D. Pantaloni. 1997. Actin depolymeriz ing factor (adf/cofilin) enhances the rate of filament turnover: Implic ation in actin-based motility. J. Cell Biol. 136:1307-1322. 61. Casella, J. F., D. J. Maack, and S. Lin. 1986. Purification and initia l characterization of a protein from skeletal musc le that caps the barbed ends of actin filaments. J. Biol. Chem. 261:10915-10921. 62. Cooper, J. A., and T. D. Pollard. 1982. Methods to measure actin polymerization. Methods Enzymol. 85:182-211. 63. Blanchoin, L., K. J. Amann, H. N. Higgs, J. B. Marchand, D. A. Kaiser, and T. D. Pollard. 2000. Direct observation of dendritic actin fila ment networks nucleated by arp2/3 complex and wasp/scar proteins. Nature. 404:1007-1011. 64. Pollard, T. D., and M. S. Mooseker. 1981. Dir ect measurement of actin polymerization rate constants by electron microscopy of actin fi laments nucleated by is olated microvillus cores. J. Cell Biol. 88:654-659. 65. Burlacu, S., P. A. Janmey, and J. Borejdo. 1992. Distribution of ac tin filament lengths measured by fluorescence microscopy. Am. J. Physiol. 262:C569-577. 66. Xu, J., J. F. Casella, and T. D. Pollard. 1999. Effect of capping protein, capz, on the length of actin filaments and m echanical properties of actin filament networks. Cell Motil. Cytoskeleton. 42:73-81. 67. Yanagida, T., M. Nakase, K. Nishiyama, and F. Oosawa. 1984. Di rect observation of motion of single F-actin filament s in the presence of myosin. Nature. 307:58-60. 68. Axelrod, D. 2001. Total internal reflec tion fluorescence microscopy in cell biology. Traffic. 2:764-774.

PAGE 150

150 69. Amann, K. J., and T. D. Pollard. 2001. Direct real-time observation of actin filament branching mediated by arp2/3 complex using total internal reflection fluorescence microscopy. Proc. Natl. Acad. Sci. U.S.A. 98:15009-15013. 70. Kuhn, J. R., and T. D. Pollard. 2005. R eal-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy. Biophys. J. 88:1387-1402. 71. Atkinson, M. A., P. K. Lambooy, and E. D. Korn. 1989. Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation. J. Biol. Chem. 264:4127-4132. 72. Pemrick, S., and A. Weber. 1976. Me chanism of inhibition of relaxation by n-ethylmaleimide treatment of myosin. Biochemistry. 15:5193-5198. 73. Amann, K. J., and T. D. Pollard. 2001. The arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments. Nat. Cell Biol. 3:306-310. 74. Xia, Y., and G. M. Whitesides. 1998. Soft lithography. Annual Review of Materials Science. 28:153-184. 75. Kumar, A., and G. M. Whitesides. 1993. Feat ures of gold having micrometer to centimeter dimensions can be formed through a combinati on of stamping with an elastomeric stamp and an alkanethiol "ink" followed by chemical etching. Appl. Phys. Lett. 63:2002-2004. 76. Kind, H., M. Geissler, H. Schmid, B. Mich el, K. Kern, and E. Delamarche. 2000. Patterned electroless deposition of copper by microcon tact printing palladi um(ii) complexes on titanium-covered surfaces. Langmuir. 16:6367-6373. 77. Nikitov, S. A., L. Presmanes, P. Ta ilhades, and D. E. Balabanov. 2002. Magnetic duplication and contact printing method. Journal of Magnetism and Magnetic Materials. 241:124-130. 78. Presmanes, L., and P. Tailhades. 2002. Fiel d assisted magnetic contact printing with soft magnetic patterned thin films. Journal of Magnetism and Magnetic Materials. 242-245:499-504. 79. Renault, J. P., A. Bernard, A. Bietsch, B. Michel, H. R. Bosshard, E. Delamarche, M. Krieter, B. Hecht, and U. P. Wild. 2003. Fabr icating arrays of singl e protein molecules on glass using microcontact printing. J. Phys. Chem. 107:703-711. 80. Xu, L., L. Robert, Q. Ouyang, F. Taddei, Y. Chen, A. B. Lindner, and D. Baigl. 2007. Microcontact printing of living bacteria arrays with cellular resolution. Nano Lett. 7:2068-2072. 81. Rozkiewicz, D. I., Y. Kraan, M. W. Werten, F. A. de Wolf, V. Subramaniam, B. J. Ravoo, and D. N. Reinhoudt. 2006. Cova lent microcontact printing of proteins for cell patterning. Chemistry. 12:6290-6297.

PAGE 151

151 82. Quist, A. P., E. Pavlovic, and S. Os carsson. 2005. Recent adva nces in microcontact printing. Anal. Bioanal. Chem. 381:591-600. 83. Theriot, J. A., J. Rosenblatt, D. A. Po rtnoy, P. J. Goldschmid t-Clermont, and T. J. Mitchison. 1994. Involvement of prof ilin in the actin-b ased motility of L. monocytogenes in cells and in cell-free extracts. Cell. 76:505-517. 84. Zeile, W. L., D. L. Purich, and F. S. Southwick. 1996. Recognition of two classes of oligoproline sequences in profilin-med iated acceleration of actin-based Shigella motility. J. Cell Biol. 133:49-59. 85. Brieher, W. M., M. Coughlin, and T. J. Mitchison. 2004. Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the arp2/3 complex. J. Cell Biol. 165:233-242. 86. Mogilner, A., and G. Oster. 2003. Force gene ration by actin polymerization II: The elastic ratchet and tethered filaments. Biophys. J. 84:1591-1605. 87. Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, and P. Cossart. 1992. L. monocytogenes-induced actin assembly requires the ActA gene product, a surface protein. Cell. 68:521-531. 88. Cameron, L. A., P. A. Giardini, F. S. Soo, and J. A. Theriot. 2000. Secrets of actin-based motility revealed by a bacterial pathogen. Nat. Rev. Mol. Cell Biol. 1:110-119. 89. Cameron, L. A., T. M. Svitkina, D. Vignj evic, J. A. Theriot, and G. G. Borisy. 2001. Dendritic organization of actin comet tails. Curr. Biol. 11:130-135. 90. Cameron, L. A., M. J. Footer, A. van Oude naarden, and J. A. Theriot. 1999. Motility of ActA protein-coated microspheres driven by actin polymerization. Proc. Natl. Acad. Sci. U.S.A. 96:4908-4913. 91. Kuo, S. C., and J. L. McGrat h. 2000. Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature. 407:1026-1029. 92. Upadhyaya, A., J. R. Chabot, A. Andreeva, A. Samadani, and A. van Oudenaarden. 2003. Probing polymerization forces by usi ng actin-propelled lipid vesicles. Proc. Natl. Acad. Sci. U.S.A. 100:4521-4526. 93. Chang, F., D. Drubin, and P. Nurse. 1997. Cdc12p, a protein require d for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J. Cell Biol. 137:169-182. 94. Kovar, D. R., and T. D. Pollard. 2004. Inser tional assembly of actin filament barbed ends in association with formin s produces piconewton forces. Proc. Natl. Acad. Sci. U.S.A. 101:14725-14730.

PAGE 152

152 95. Pruyne, D., M. Evangelista, C. Yang, E. Bi, S. Zigmond, A. Bretscher, and C. Boone. 2002. Role of formins in actin assembly : Nucleation and barbed-end association. Science. 297:612-615. 96. Zigmond, S. H., M. Evangelista, C. Boone, C. Yang, A. C. Dar, F. Sicheri, J. Forkey, and M. Pring. 2003. Formin leaky cap allows elongation in the presence of tight capping proteins. Curr. Biol. 13:1820-1823. 97. Mogilner, A., and G. Oster. 1996. Cell motility driven by actin polymerization. Biophys. J. 71:3030-3045. 98. Peskin, C. S., G. M. Odell, and G. F. Oster. 1993. Cellular motions and thermal fluctuations: The Brownian ratchet. Biophys. J. 65:316-324. 99. Hill, T. L. 1981. Microfilament or microtubu le assembly or disassembly against a force. Proc. Natl. Acad. Sci. U.S.A. 78:5613-5617. 100. Abraham, V. C., V. Krishnamurthi, D. L. Taylor, and F. Lanni. 1999. The actin-based nanomachine at the leading edge of migrating cells. Biophys. J. 77:1721-1732. 101. Kovar, D. R., J. Q. Wu, and T. D. Po llard. 2005. Profilin-mediated competition between capping protein and formin cdc12p duri ng cytokinesis in fission yeast. Mol. Biol. Cell. 16:2313-2324. 102. Romero, S., D. Didry, E. La rquet, N. Boisset, D. Pantalon i, and M. F. Carlier. 2007. How ATP hydrolysis controls filament assembly from profilin-actin: Implication for formin processivity. J. Biol. Chem. 282:8435-8445. 103. Kwiatkowski, A. V., F. B. Gertler, and J. J. Loureiro. 2003. Function and regulation of Ena/VASP proteins. Trends Cell Biol. 13:386-392. 104. Geese, M., K. Schluter, M. Rothkegel, B. M. Jockusch, J. Wehland, and A. S. Sechi. 2000. Accumulation of profili n II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed. J. Cell Sci. 113 (Pt 8):1415-1426. 105. Clemmens, J., H. Hess, R. Lipscomb, Y. Hane in, K. F. Bohringer, C. M. Matzke, G. D. Bachand, B. C. Bunker, and V. Vogel. 2003. Mechanisms of microtubule guiding on microfabricated kinesin-coated surfaces: Ch emical and topographic surface patterns. Langmuir. 19:10967-10974. 106. Pardee, J. D., and J. A. Spudich 1982. Purification of muscle actin. Methods Enzymol. 85 Pt B:164-181. 107. Bernard, A., E. Delamarche, H. Schmid, B. Michel, H. R. Bosshard, and H. Biebuyk. 1998. Printing patterns of proteins. Langmuir. 14:2225-2229. 108. Landau, L. D., and E. M. Lifshitz. 1986. Theory of elasticity. Reed Educational and Professional Publishing Ltd., New York.

PAGE 153

153 109. Ostap, E. M., T. Yanagida, and D. D. Thomas. 1992. Orientational distribution of spin-labeled actin oriented by flow. Biophys. J. 63:966-975. 110. Lee, C. S., S. H. Lee, S. S. Park, Y. K. Kim, and B. G. Kim. 2003. Protein patterning on silicon-based surface using b ackground hydrophobic thin film. Biosens. Bioelectron. 18:437-444. 111. Welch, M. D., J. Rosenblatt, J. Skoble D. A. Portnoy, and T. J. Mitchison. 1998. Interaction of human arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science. 281:105-108. 112. Plastino, J., I. Lelidis, J. Prost, a nd C. Sykes. 2004. The effect of diffusion, depolymerization and nucleation promoting factors on actin gel growth. Eur. Biophys. J. 33:310-320. 113. Zeile, W. L., F. Zhang, R. B. Dickinson, and D. L. Purich. 2005. Listeria's right-handed helical rocket-tail trajectories: Mechanistic im plications for force generation in actin-based motility. Cell Motil. Cytoskeleton. 60:121-128. 114. Welch, M. D., A. Iwamatsu, and T. J. M itchison. 1997. Actin polym erization is induced by arp2/3 protein complex at the surface of Listeria monocytogenes. Nature. 385:265-269. 115. Loisel, T. P., R. Boujemaa, D. Pantaloni, and M. F. Carlier. 1999. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature. 401:613-616. 116. Cameron, L. A., J. R. Robbins, M. J. Footer, and J. A. Theriot. 2004. Biophysical parameters influence actin-based movement, tr ajectory, and initiation in a cell-free system. Mol. Biol. Cell. 15:2312-2323. 117. Oudenaarden, A., and J. A. Theriot. 1999. Cooperative symmetry-breaking by actin polymerization in a model for cell motility. Nat. Cell Biol. 1:493-499. 118. Brown, S. S., and J. A. Spudich. 1979. Nu cleation of polar actin filament assembly by a positively charged surface. J. Cell Biol. 80:499-504. 119. Vignjevic, D., D. Yarar, M. D. Welch, J. Peloquin, T. Svitkina, and G. G. Borisy. 2003. Formation of filopodia-like bundles in vitro from a dendritic network. J. Cell Biol. 160:951-962. 120. Rogers, J. A., K. E. Paul, and G. M. Wh itesides. 1998. Quantifying distortions in soft lithography. Journal of Vacuum Science and Technology B. 16:88-97.

PAGE 154

154 BIOGRAPHICAL SKETCH K imberly A. Interliggi was born and raised in Canton, Ohio. She graduated from Jackson High School in 2000 and attended Ohio University on a full-tuition scholarship. She received her BSChe (magna cum laude) in June 2003. In August 2003, she enrolled in the PhD program in the Department of Chemical E ngineering at the University of Florida. She began working for Dr. Richard Dickinson in January 2004 and rece ived her doctorate in December 2007.