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Shigella flexneri Recruits Host Cell Myosin-X for Efficient Formation of Filopodia

Permanent Link: http://ufdc.ufl.edu/UFE0021701/00001

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Title: Shigella flexneri Recruits Host Cell Myosin-X for Efficient Formation of Filopodia
Physical Description: 1 online resource (69 p.)
Language: english
Creator: Bishai, Ellen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: Shigella flexneri is a gram negative rod that causes Shigellosis, a highly communicable disease responsible for significant morbidity and mortality in underdeveloped countries. Shigella is able to enter the cytoplasm of gastrointestinal epithelial cells, and subsequently usurp host cell actin to move intracellularly and to the cell periphery to form finger-like filopodia. These protrusions are ingested by neighboring cells, allowing Shigella to spread from cell to cell and produce hemorrhagic plaques. Much work has been done to delineate the factors and mechanisms necessary for Shigella-induced actin-based intracellular motility. In contrast, the factors and mechanisms underlying filopodia formation by this bacterium are not well characterized. The unconventional myosin, myosin X, has recently been shown to contribute to filopodia formation in various mammalian cell lines (Berg, 2002). We found that in living HeLa cells, GFP-Myosin X concentrates along the sides of filopodia containing Shigella. Immunofluorescence microscopy utilizing a specific anti-myosin X antibody corroborated these findings. Furthermore, Listeria monocytogenes, another intracellular pathogen that usurps host cell actin for motility, fails to recruit GFP-Myosin X. Knocking down endogenous myosin X levels in Shigella-infected cells using myosin X-specific siRNA results in approximately a 30% reduction in Shigella-induced filopodia formation when compared to cells containing native levels of myosin X (avg 11.01?m compared to 14.73?m, respectively; p=0.0005). Additionally, transfection of living HeLa cells with a GFP-Myosin X-HMM construct (contains only head, neck, and coiled coil domains of myosin X) resulted in a significant reduction in the lengths of Shigella-induced filopodia when compared to cells transfected with full-length GFP-Myosin X (avg 11.66?m compared to 15.25?m, p = 0.006). We conclude that myosin X is an important component in filopodia formation by Shigella flexneri and that the tail region is responsible for conveying this function.
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 Ellen Bishai.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Southwick, Frederick S.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
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Permanent Link: http://ufdc.ufl.edu/UFE0021701/00001

Material Information

Title: Shigella flexneri Recruits Host Cell Myosin-X for Efficient Formation of Filopodia
Physical Description: 1 online resource (69 p.)
Language: english
Creator: Bishai, Ellen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Shigella flexneri is a gram negative rod that causes Shigellosis, a highly communicable disease responsible for significant morbidity and mortality in underdeveloped countries. Shigella is able to enter the cytoplasm of gastrointestinal epithelial cells, and subsequently usurp host cell actin to move intracellularly and to the cell periphery to form finger-like filopodia. These protrusions are ingested by neighboring cells, allowing Shigella to spread from cell to cell and produce hemorrhagic plaques. Much work has been done to delineate the factors and mechanisms necessary for Shigella-induced actin-based intracellular motility. In contrast, the factors and mechanisms underlying filopodia formation by this bacterium are not well characterized. The unconventional myosin, myosin X, has recently been shown to contribute to filopodia formation in various mammalian cell lines (Berg, 2002). We found that in living HeLa cells, GFP-Myosin X concentrates along the sides of filopodia containing Shigella. Immunofluorescence microscopy utilizing a specific anti-myosin X antibody corroborated these findings. Furthermore, Listeria monocytogenes, another intracellular pathogen that usurps host cell actin for motility, fails to recruit GFP-Myosin X. Knocking down endogenous myosin X levels in Shigella-infected cells using myosin X-specific siRNA results in approximately a 30% reduction in Shigella-induced filopodia formation when compared to cells containing native levels of myosin X (avg 11.01?m compared to 14.73?m, respectively; p=0.0005). Additionally, transfection of living HeLa cells with a GFP-Myosin X-HMM construct (contains only head, neck, and coiled coil domains of myosin X) resulted in a significant reduction in the lengths of Shigella-induced filopodia when compared to cells transfected with full-length GFP-Myosin X (avg 11.66?m compared to 15.25?m, p = 0.006). We conclude that myosin X is an important component in filopodia formation by Shigella flexneri and that the tail region is responsible for conveying this function.
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 Ellen Bishai.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Southwick, Frederick S.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021701:00001


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,Nlihgell, flexneri RECRUITS HOST CELL MYOSIN-X FOR EFFICIENT FORMATION OF
FILOPODIA




















By

ELLEN A BISHAI


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 Ellen A Bishai

































To God, without whose hand this would not have been possible; and to my family whose love
and prayers have always provided a fervent base of support.









ACKNOWLEDGMENTS

As I already stated, I would like to thank my family for all their love, prayers, and

support throughout my life and these last few years. I am also greatly indebted to my mentor

who was always a wonderful source of encouragement and motivation. I honestly wouldn't have

made it through this experience without his continual guidance and support. Lastly, but

definitely not least, I would like to thank all of my lab-mates, past and present. Not only did they

provide me with technical guidance when it was needed, but their friendship was a valuable

resource in and of itself.









TABLE OF CONTENTS

page

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

L IST O F T A B L E S ...................................................................................................... . 7

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

A B S T R A C T ................................ .................. .......................... ................ .. 9

CHAPTER

1 INTRODUCTION ............... ..................................................... ..... 11

Significance and Epidemiology .......................... ............................................11
Filopodium Formation in Eukaryotic Cells........................... ..................................... 18
M yosins and Filopodia Form ation ............................................................................ ... .... 20
Overview of M yosins ........................ ............ ............... ......... .... .......20
Myosin-X (M10) and Filopodia Formation ......................................... ...............21

2 M E TH O D S A N D M A TER IA L S ........................................ .............................................27

Cell Infections with S. flexneri and M10 Localization............................ ...............27
Cell Cultures and Bacterial Strains .............. ..................................... ...............27
Transfection with cDNA Plasmids ........... ..... ......... ................... 27
C ell In v asio n ................................................. ......................... ... ...... 2 8
Immunofluorescence and Phalloidin Stain ....................................... ...............28
LY 294002 Treatm ent .......................................... .. .. ........... .... ....... 29
V ideo M icroscopy ................. .................................... .. ........ .. .............29
T ransfection w ith siR N A 's............................................................................ ................... 29
W western B lots.....................................................30
P laq u e A ssay s ............................................................................... 3 0

3 R E SU L T S .............. ... ................................................................32

M 10 Localizes to S. flexneri Contained in Filopodia ........................................ ............ 32
In tro d u ctio n ........................................ ....... ... ......... ........ .... ......................3 2
M10 localizes to motile .1/Vge/ll, but not motile Listeria in HeLa cells......................33
Tropomyosin localizes to Listeria-Induced, but not .\/ige/Al-Induced, Actin Tails .......34
Tail Region of M10 Facilitates ./lige//, t's Ability to Form Longer Filopodia .......................34
In tro du ctio n ................................ ................................... .................. ....... ..................... 3 4
Absence of M10 Tail Region Results in Shorter .\/igell, -Induced Filopodia .............36
The MyTH4 and FERM Domains of M10 Are Not Required for Efficient /lge/lt-
Induced Filopodium Formation .........................................................37
Inhibition of PI3K products and overexpression of GFP-Akt-PH Do Not Affect
.\l/ige/hl -Induced Filopodial L engths ................................................... ............... 38









Reduction of Endogenous M10 Levels Curtails S. flexneri Cell-to-Cell Spread in HeLa
C ell M onolayers In Vitro .......................................................................... ....................40

4 D IS C U S S IO N ......................................... .. ......................................................5 4

M 10 Recruitm ent to .\lige// flexneri .......... .. .......................................... ....................54
M10 Contribution During .\l/igel/-induced Filopodium Formation............................. 56
Involvement of M1O's Tail Region in .\/nlge//A-Induced Filopodium Formation .................57
F u tu re D ire ctio n s .............................................................................................................. 5 8

L IST O F R EFE R E N C E S ...................... ....................................................................60

B IO G R A PH IC A L SK E T C H .............................................................................. .....................69









































6









LIST OF TABLES


Table page

1-1 List of S. flexneri TTSS effector proteins and their functions..............................25

1-2 List of cytoskeletal components found in mammalian cells...........................................26









LIST OF FIGURES


Figure page

3-1A Time-lapse pictures of GFP-M10 localizing to motile intracellular Shigella in PtK2
c e lls ....................................................................................... 4 1

3-1B Time lapse pictures of PtK2 cells transfected with GFP-M10 and infected with
L isteria ......................................................... ..................................4 2

3-2 M10 antibody localizes to intracellular Shigella and Shigella-laden filopodia ...............43

3-3 Tropomyosin monoclonal antibody localizes to Listeria-, but not Shigella-induced
actin tails in H eL a cells........... ................... ........ ........... .. ............. 44

3-4 The GFP-tagged cDNA constructs used in cell transfections ............... .....................45

3-6 Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-HMM,
infected with Shigella, and analyzed using video microscopy. .......................................46

3-7A Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-AFERM,
infected with Shigella, and analyzed using video microscopy. .......................................47

3-7B Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-
AMyTH4AFERM, infected with Shigella, and analyzed using video microscopy ..........48

3-8A HeLa cells were infected with Shigella, treated with either DMSO (control) or LY
294002, and analyzed using video microscopy. ..................................... ............... 49

3-8B HeLa cells were either transfected with GFP-Akt-PH or not transfected (control),
infected with Shigella, and analyzed using video microscopy. .......................................50

3-8C HeLa cells were either transfected with GFP-PLC6-PH or GFP only, infected with
\/ngel//l, and analyzed using video microscopy. .................................... .................51

3-9 Bar graph showing number of plaques formed on HeLa cell monolayers by S.
f lex n eri ......................................................... .................................. 5 2

3-10 HeLa cells were either transfected with non-targeting siRNA (Ctrl siRNA) or M10
siRNA, infected with .\/ngel//i, and analyzed using video microscopy ............................53









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
.\l/ge//u'flexneri RECRUITS HOST CELL MYOSIN-X FOR EFFICIENT FORMATION OF
FILOPODIA

By

Ellen Bishai

December 2007

Chair: Frederick S. Southwick
Major: Medical Sciences Immunology and Microbiology

hg, i/, iflexneri is a gram negative rod that causes Shigellosis, a highly communicable disease

responsible for significant morbidity and mortality in underdeveloped countries. Shigella is able to enter

the cytoplasm of gastrointestinal epithelial cells, and subsequently usurp host cell actin to move

intracellularly and to the cell periphery to form finger-like filopodia. These protrusions are ingested by

neighboring cells, allowing \/iig 1 to spread from cell to cell and produce hemorrhagic plaques. Much

work has been done to delineate the factors and mechanisms necessary for \/i,g, /,-induced actin-based

intracellular motility. In contrast, the factors and mechanisms underlying filopodia formation by this

bacterium are not well characterized. The unconventional myosin, myosin X, has recently been shown to

contribute to filopodia formation in various mammalian cell lines (Berg, 2002). We found that in living

HeLa cells, GFP-Myosin X concentrates along the sides of filopodia containing \N/giL i/.

Immunofluorescence microscopy utilizing a specific anti-myosin X antibody corroborated these findings.

Furthermore, Listeria monocytogenes, another intracellular pathogen that usurps host cell actin for

motility, fails to recruit GFP-Myosin X. Knocking down endogenous myosin X levels in \'/ig l i-

infected cells using myosin X-specific siRNA results in approximately a 30% reduction in h, i g, /i-

induced filopodia formation when compared to cells containing native levels of myosin X (avg 11.01 m

compared to 14.73gm, respectively; p=0.0005). Additionally, transfection of living HeLa cells with a

GFP-Myosin X-HMM construct (contains only head, neck, and coiled coil domains of myosin X) resulted









in a significant reduction in the lengths of \ihg //, -induced filopodia when compared to cells transfected

with full-length GFP-Myosin X (avg 11.66(m compared to 15.25.im, p = 0.006). We conclude that

myosin X is an important component in filopodia formation by \/hig, I/a flexneri and that the tail region is

responsible for conveying this function.









CHAPTER 1
INTRODUCTION

Significance and Epidemiology

Shigellosis, also known as acute bacillary dysentery, is caused by .\/ige/l/ spp. and

annually affects 164 million people worldwide and causes 1.1 million deaths with the majority of

cases occurring in children living in developing countries (Jennison and Verma, 2004). The

disease is characterized by loose stools containing blood and pus as well as fever, abdominal

cramps, and tenesmus (an incomplete sense of evacuation with rectal pain) (Sur etal., 2004).

The disease is highly communicable and is spread via the fecal-oral route, which can be

facilitated by poor water sanitation, poor hygiene, and close personal contact. The higher

incidence of disease spread in developing countries is often attributed to these conditions

(Jennison and Verma, 2004). Countries where epidemics have been reported include South

American countries, Asian countries (i.e., Bangladesh, Sri Lanka, Maldives, Nepal, Bhutan, and

Myanmar), and regions in southern and eastern India (Sur et al., 2004). Although the disease

primarily affects populations living in developing countries, travelers visiting endemic areas may

also be infected if they do not take proper precautionary hygienic measures (Sur et al., 2004).

Additionally, in the United States, outbreaks that originate in day care centers are not uncommon

and can subsequently spread throughout communities (Shane et al., 2003).

The etiological agents of Shigellosis belong to the genus .\/ilg//At and include four

species: S. dysenteriae (16 serotypes, of which serotype 1 is the most deadly due to its ability to

produce shiga toxin), S. flexneri (6 serotypes, most of which are responsible for endemic disease

in developing countries with serotype 2a being the most prevalent), S. boydii (8 serotypes) and S.

sonnei (1 serotype, commonly the source of day care center outbreaks) (Sansonetti 2001). Being









endemic in most developing countries, S. flexneri has been reported to cause more mortality than

any other ./lge//At species (Bennish and Wojtyniak, 1991; Kotloff et al., 1999).

Pathogenesis of S. flexneri

.\/ge//ll flexneri is a gram-negative, nonsporulating, facultative anaerobic rod that causes

an invasive infection of the human colon. The key factors involved for successful pathogenesis

of S. flexneri include 1) the ability to traverse the colonic epithelium; 2) the ability to induce

uptake into the non-phagocytic cells of the colonic epithelium; and 3) the ability to usurp the host

cell's actin machinery in order to facilitate intra- and intercellular motility, which result in the

spreading of the infection (Sansonetti 2001).

Colonic Epithelium: Invasion and Entry

Once ingested, S. flexneri is able to pass through the stomach unharmed. This attribute

enables S. flexneri to successfully establish an infection even if only a small number of

microorganisms is ingested (as few as 10-100 microorganisms) (Sansonetti 2001). The reason

why only a small number of microorganisms is sufficient to establish an infection is believed to

be the presence of acid-resistance pathways possessed by the bacteria that are induced upon

encountering hostile acidic environments such as the human stomach (Gianella et al., 1972;

Jennison and Verma, 2007). The two pathways thought to play a role in .\/lge/l/ acid-resistance

are the acid-resistance pathway 1 (AR1) a stationary-phase, acid-induced, glucose-repressed

oxidative pathway and the acid-resistance pathway 2 (AR2) a stationary-phase, glutamate-

dependent acid-resistance (GDAR) pathway. When bacteria reach the intestines, they are then

able to traverse the colonic epithelium by way of M cells (Sansonetti and Phalipon, 1999). At

the underlying lamina propria, bacteria go on to invade macrophages. After engulfment, S.

flexneri is able to lyse the phagocytic vacuole and escape into the macrophage cytoplasm. There,

.\/lge//lt release the effector protein IpaB, which triggers apoptosis via its ability to bind









interleukin-lp converting enzyme (ICE), also known as Caspase-1 (Zychlinksi et al., 1992; Chen

et al., 1996). Upon induction of apoptosis, IL-10 is released which, in turn, recruits more

macrophages and dendritic cells to the site of infection (Cossart and Sansonetti, 2004). IL-10 is

also a potent recruiter of polymorphonuclear leukocytes (PMNs). When PMNs arrive at the

infection site, they transmigrate through the colonic epithelial tissue to the basolateral surface.

This process causes major tissue destruction and results in the formation of hemorrhagic plaques

or lesions as well as serving to further spread the zone of infection (Sansonetti, 2001). After

escaping from macrophages, S. flexneri can then invade intestinal epithelial cells from the

basolateral side by inducing cell ruffling using its type III secretion system (TTSS).

.\/ge//At's TTSS is encoded by the mxi-spa region of the virulence plasmid (Espina et al.,

2006). From "inside-out", the apparatus is comprised of a cytoplasmic bulb and a disk-like

structure that spans the bacterium's inner and outer membranes. A needle like structure crosses

these domains and extends outside the outer membrane. The needle structure mediates the

delivery of several effector proteins directly from the bacterial cytoplasm into the host cell

cytoplasm. The proteins that comprise and are also transferred through .\/lge//ll's TTSS have

been identified and include IpaB, IpaC, IpaD, IpaA, IpgD, and VirA (Table 1-1). IpaB and IpaC

form an "Ipa complex" at the host cell membrane surface, which has been shown to be sufficient

for invasion into host cells (Menard et al., 1996; Table 1-1). Insertion of the IpaBC complex into

the host cell membrane creates a pore and activates signal transduction pathways that are

responsible for generating membrane ruffles. The IpaBC complex is also known to be necessary

for enabling \/Nige/ll escape from the phagocytic vacuole in macrophages and host cells (Page et

al., 1999). IpaB has been shown to bind to the cell surface protein CD44 and associate with

as51-integrin (Lafont et al., 2002). IpaB also eventually leads to induction of apoptosis of









.\s/ge//Al-infected macrophages. Besides associating with IpaB to create the IpaBC complex that

is necessary for entry into epithelial cells and subsequent lysis of the phagocytic vacuole, IpaC

also acts to trigger F-actin nucleation via its C-terminal domain (Tran Van Nhieu et al., 1999).

IpaA helps to mature the entry focus induced by .\/nlgel// Once injected into the cell, it binds the

actin related protein vinculin. In mammalian cells, vinculin is usually found at sites of focal

adhesion and is known to act as a linker between actin filaments and the plasma membrane (Tran

Van Nhieu et al., 1997). Normally, vinculin exists in an autoinhibitory state in the cell due to an

intramolecular association between the 95 kDa head and the 30 kDa tail domains. In this

conformation, vinculin's F-actin binding site contained within the tail domain is masked and,

therefore, unavailable to bind actin filaments (Johnson and Craig, 1995). While under normal

conditions, an external growth hormone signal would be required to alleviate vinculin's

autoinhibitory state, in 1999 Bourdet-Sicard and colleagues were able to show that binding of

IpaA to vinculin is able to relieve its autoinhibitory state as well as enhance its ability to interact

with F-actin. They were also able to show that the resulting complex depolymerized actin

filaments both in vitro and in microinjected cells. This is in some opposition to the role usually

ascribed to vinculin in mammalian cells in which it is thought that vinculin recruits VASP in cell

focal adhesion sites, which can, in turn, promote actin filament assembly (Holt et al., 1998).

Nevertheless, during \/ngel//t infection, the vinculin-IpaA complex can act to depolymerize actin

filaments in a controlled manner at the site of bacterial entry, forming a phagocytic cup

underneath the bacterium and allowing filopodial structures to extend around the bacterium,

facilitating its engulfment (Bourdet-Sicard et al., 1999).

IpaD is an effector protein known to be important in regulating secretion of other Ipa

proteins as well as forming polymers with IpaC. This complex has been shown to be required for









binding and entry of ./ngel//t (Picking et al., 2005). Another effector protein that has been

implicated in binding and entry of .\/ngel//t is IpgD (Niebuhr et al., 2000). This protein exhibits

two motifs that are present in mammalian inositol polyphosphate 4-phosphatase and upon

secretion, it specifically dephosphorylates phosphatidylinositol (4,5) bis-phosphate [PI(4,5)P2] to

yield PI(5)P. PI(4,5)P2 is known to enable membrane-cytoskeletal interactions via its presence

in the cytosolic face of the host membrane and its ability to bind several actin regulatory proteins

(Czech, 2000). Therefore, IpgD's action is thought to assist in relaxing membrane-cytoskeleton

interactions in order to further facilitate the extension of actin filaments during the entry process

(Niebuhr et al., 2002). Indeed, Niebuhr and colleagues were able to show that expression of

IpgD in mammalian cells led to a strong decrease in tether force presumably by uncoupling of

the plasma membrane from the underlying actin cytoskeleton.

Shortly after being taken up by the host cell, S. flexneri is able to escape the harsh

environment of the phagolysosome by inducing lysis of the vacuole and escaping into the

cytosol. Epithelial cells that have been invaded release IL-8, which, in turn, recruits neutrophils

to the site of infection. It is the presence of neutrophils that results in pus being passed in the

stool, while the inflammation that ensues at the infected site causes actual damage to the

intestinal epithelium, resulting in bloody diarrhea (Niyogi, 2005).

Actin-based Intra- and Intercellular Motility

Aside from causing damage to the intestinal epithelium, S. flexneri possesses another

attribute that contributes to successful pathogenesis. Upon escaping the vacuole and being

released into the cytoplasm, S. flexneri goes on to usurp the host cell's actin machinery to form

rocket tails which propel the bacteria within the cell and also allows direct spread to neighboring

cells without being exposed to any extracellular milieu. This aspect of the pathogenesis process









plays an important role in allowing the infection to spread without detection by macrophages or

antibodies (Finlay and Falkow, 1997).

Normally in cells, actin dynamics are meticulously controlled by an array of proteins in

response to specific extracellular or intracellular stimuli (Pantaloni et al., 2001). Some key

components involved in normal actin dynamics in the cell include monomeric and filamentous

actin, Arp2/3, profilin, thymosin 0-4, N-WASP (neuronal Wiskott-Aldrich syndrome protein),

VASP (vasodilator-stimulated phosphoprotein), Rho family proteins, ADF/cofilin, and capping

proteins. In non-muscle cells, the actin cytoskeleton plays key roles in whole-cell motility

(Pollard and Borisy, 2003) and trafficking of intracellular organelles (Engqvist-Goldstein and

Drubin, 2003).

Actin exists in two forms in the cell: monomeric G-actin and filamentous F-actin. The

Arp2/3 complex, which consists of the actin related proteins Arp2, Arp3, and five other subunits,

is vital in nucleating G-actin to begin F-actin formation. In order to initiate nucleation, the

Arp2/3 complex must first be activated by WASP (Wiskott-Aldrich syndrome protein) or N-

WASP. N-WASP exists in an autoinhibitory state in the cell due to sequences near the N-

terminal region interacting with sequences in the C-terminal region (Bompard and Caron, 2004).

In this conformation, binding domains are unavailable, rendering N-WASP unable to interact

with binding partners. It is activated and recruited to the cell membrane via activated members

of the Rho family small GTPases such as Cdc42 and Rac. Once recruited to the cytosolic face of

the plasma membrane, N-WASP acts as a scaffolding protein that brings Arp2/3 and G-actin into

spatial and functional proximity. In its unfolded state, N-WASP's C-terminal VCA domain is

made available for Arp2/3 and G-actin to bind. Nucleation by Arp2/3 leads to the formation of

polarized F-actin filaments. The end where new monomers are added is termed the barbed (or









plus) end, while the end from which "older" monomers dissociate is termed the pointed (or

minus) end. The terms "barbed" and "pointed" refer to previous observations of actin filaments

decorated with myosin II S1 fragments (these fragments contain only the head and neck region

of myosin II). In these experiments, the S1 fragments bind the actin filament at a 450 angle when

ATP is absent, creating the appearance of a barbed end and a pointed end (Momet et al., 1981).

The protein profilin binds G-actin monomers, ushers them onto the barbed end of the growing

actin filament, and catalyzes their incorporation into the filament by enhancing ATP-ADP

exchange on monomers (Pollard, 2007). N-WASP and VASP contain polyproline regions to

which profilin can bind (Kang et al., 1997; Suzuki and Sasakawa, 2001). It has been proposed

that VASP's role is to bind the growing F-actin chain and bring in profilin-actin complexes to the

barbed end, aiding in efficient filament elongation (Krause et al., 2003). In order to orchestrate

directional motility, capping proteins are needed to stop the growth of one or more filaments so

that others may be allowed enhanced growth to propel the cell in the desired direction. Lastly,

ADF/cofilin is a protein that binds to the sides of actin filaments then severs and caps the

filament, enhancing disassembly at the barbed ends.

In the case of bacterial invasion, however, S. flexneri and a handful of other pathogens have

each devised distinct ways of activating the host cell's actin machinery without the involvement

of external or internal signals. Specifically, S. flexneri expresses an outer-membrane protein,

IcsA (or VirG), which is necessary and sufficient (Goldberg and Theriot, 1995; Bernardini et al.,

1989) for initiating its actin-based motility. IcsA is responsible for binding the host protein, N-

WASP (Loisel et al., 1999). Binding of IcsA is able to relieve the autoinhibition of N-WASP in

a Cdc42-independent manner. The open conformation of N-WASP in turn activates the Arp2/3

complex, which is required to first stimulate nucleation of actin filament assembly at the









bacterial surface (Gouin et al., 2005). Actin filaments are assembled at one pole of the bacterial

surface with the barbed end of the filament facing the bacterium. Subsequent addition of ATP-

bound monomers to the barbed end of filamentous actin generates the force necessary to propel

the bacterium throughout the cytosol (Gouin et al., 2005). Using this mode of motility, S.

flexneri is able to move throughout the host cell cytosol until it reaches the inner face of the

plasma membrane. Once there it is then able to generate the formation of finger-like protrusions

called filopodia. These structures harbor a bacterium at their tips and extend from the infected

cell into an uninfected neighboring cell, which subsequently engulfs and ingests the bacteria-

containing structure (Sansonetti et al., 1994). Once engulfed, the bacterium is able to lyse the

double membrane vacuole that contains it by way of TTSS effector proteins and the cycle is

repeated again (Allaoui et al., 1992). Therefore, it is this process that confers upon S. flexneri

the ability to rapidly spread infection along the colonic epithelium. Further, it has been shown S.

flexneri that are defective in implementing this process are unable to set up successful infections

in animal models (Sansonetti et al., 1991). Despite the significance of this facet of S. flexneri's

pathogenesis, little is understood regarding which factors) and/or processes are required for

forming filopodia once bacteria reach the host cell periphery (Pust et al., 2005).

Filopodium Formation in Eukaryotic Cells

Filopodia are defined as actin-rich organelles that extend from the cell front in migrating

cells. They play a key role in exploring new space during cell migration, as well as subsequently

mediating adhesion to extracellular matrices or cells. Furthermore, several protrusion structures

in specialized cells namely, lymphocyte and brush border microvilli as well as stereocilia on

cochlear cells have been proposed to be related to filopodia (Faix and Rottner, 2006). While it

is currently unknown whether filopodia are initiated as independent structures or if they are

borne of preexisting cell membrane protrusions called lamellipodia, in recent years a number of









molecular components have been identified that are associated with filopodia formation. One of

the main components capable of potently inducing filopodium formation is Cdc42, a member of

the Rho-family GTPases (Nobes and Hall, 1995). This phenomenon is thought to occur via

direct interaction of Cdc42 with N-WASP, which is a known activator of the Arp2/3 complex

(Stradal et al., 2004). However, although this complex may be important for nucleation of actin

filaments leading to initiation of filopodium protrusion, the Arp2/3 complex is altogether absent

from the tips of filopodia, indicating that further elongation requires the contribution of other

components (Faix and Rottner, 2006). Proteins of interest that have been shown to be present at

filopodial tips include N-WASP, formins, members of the Ena/VASP family, fascin, and insulin

receptor substrate p53 (IRSp53).

Like N-WASP, IRSp53 has been shown to be an effector protein downstream of Cdc42

(Govind et al., 2001). Of particular interest, it has been proposed to contribute to efficient

filopodium formation through its interaction with members of the Ena/Mena/VASP family

(Krugmann et al., 2001). Krugmann and colleagues (2001) were able to show that this

interaction is brought about when activated Cdc42 binds to a partial CRIB motif found within the

central region of IRSp53. Binding of Cdc42 prevents an intramolecular interaction within

IRSp53 and, thereby, allows the recruitment of Mena to the IRSp53 SH3 domain. Previously, it

has been proposed that VASP's presence at filopodial tips may serve to uncap the barbed ends of

actin filaments, thereby encouraging monomer addition and filopodium elongation (Bear et al.,

2002; Krause et al., 2003). More recently, however, studies have shown that VASP's important

role in filopodial formation relies heavily on its F-actin bundling ability. Furthermore, these

studies have also suggested that VASP does not act as a competitor to capping proteins nor does

it depolymerize from F-actin barbed ends (Schirenbeck et al., 2005; Schirenbeck et al., 2006).









However, although VASP may play an important role in facilitating filopodium elongation, it

does not have the inherent ability to drive filopodium protrusion. A family of proteins, termed

diaphanous-related formins (Drf), has been shown to be involved in this facet of filopodia

formation. These proteins are characterized by a highly conserved domain called the formin

homology 2 (FH2) domain. This domain has been shown to possess the ability to nucleate and

elongate unbranched actin filaments (Wallar and Alberts, 2003), creating parallel networks of

actin filaments as opposed to a network in which filaments "branch" off one another at angles of

700. This characteristic is also in line with the observation that the Arp2/3 complex, which is

primarily known to nucleate the branched actin filament networks mentioned above, is not found

throughout the length or at the tips of filopodia. Furthermore, Drf s are also known to be

downstream effector proteins of Cdc42 (Peng et al., 2003). In addition to protrusion, another

aspect that is important in maintaining filopodial integrity is the action of stabilization of the

parallel actin filaments that constitute the filopodium. The actin-binding protein fascin serves

this need by cross-linking parallel filopodial actin filaments. Fascin is proposed to contain two

actin-binding sites, one of which has been identified, that confer its ability to bundle filopodial

actin filaments and thereby provide structural stability to the filopodium (Kureishy et al., 2002).

Myosins and Filopodia Formation

Myosins comprise a large superfamily of actin-dependent molecular motor proteins that

perform a variety of functions in the cell. Recent research suggests they are also involved in

proper formation of filopodia and similar structures.

Overview of Myosins

There are currently 15 distinct classes of myosins; they typically contain three fundamental

domains that designate them as members of the myosin superfamily: 1) a motor domain (also

called a head domain) that interacts with actin and binds ATP, 2) a neck domain that is able to









bind light chains or calmodulin, and 3) a tail domain that binds specific cargo(s) and/or positions

the motor domain so that it can interact with actin (Sellers, 2000). Among the three domains, the

motor domain is the most conserved across the 15 classes, while the neck domain and the tail

domain can vary slightly and widely, respectively. Myosins can exist as single molecules or as

homodimers. The possibility of dimerization is usually dependent upon the presence of a coiled

coil region in the neck domain. There is no predetermined criteria stating which regions)

constitutes a myosin tail domain. Across the 15 classes, this domain is the most divergent and is

usually the domain that confers upon each class of myosin its unique functionality.

Class II myosins are predominantly functional in muscle cells and were the first class of

myosins to be identified. For several decades they were the only class thought to exist and it is

for this reason that they are sometimes referred to as "conventional" myosins (Sellers, 2000).

Hence, all myosin classes that were discovered subsequently are referred to as "unconventional"

myosins. The functions of unconventional myosins are manifested in many cellular processes

including membrane trafficking, cell movements, and signal transduction (Mermall et al., 1998).

It has even been shown that certain myosins play a key role in several sensory systems, including

hearing, balance, and vision. Of note is the finding that myosins Ic, VI, VIIa, XVa, and others

are necessary for the proper formation of stereocilia (microvilli-like cell projections found on

hair cells in the inner ear). Mutations in these genes have been shown to be the cause of hearing

loss and some balance disorders in vertebrates and Drosophila melanogaster (Frolenkov et al.,

2004). This is one specific example that illustrates the involvement of myosins in the proper

formation and/or extension of actin-rich protrusions.

Myosin-X (M10) and Filopodia Formation

Myosin-X (M10) is a recently discovered unconventional myosin that is found

ubiquitously, though in low quantities, in various tissue types (Berg et al., 2000). Although its









structure has not yet been determined, M10 is proposed to be a double-headed myosin motor due

to the presence of a putative coiled coil domain. Each tail domain contains three total pleckstrin

homology (PH) domains (only two of which are thought to be functional), a myosin tail

homology 4 (MyTH4) domain, and a band 4.1/ezrin/radixin/moesin (FERM domain). This

unique tail composition makes it the founding and sole member of its class (Sellers et al., 2000).

In 2002, Berg and colleagues were able to show that overexpression of full-length M10, but not

M10 truncates, was able to increase the number and length of filopodia formed in HeLa cells.

Furthermore, by using a GFP-M10 construct, they were also able to demonstrate that M10

specifically localizes to the tips of filopodia and undergoes forward and rearward movement

within the filopodia. This observation led them to suggest that M10 acts as an intrafilopodial

motor that may have a role in delivering cargo(s) to filopodial tips. While the issue of whether

or not M10 does in fact play a role in transporting cargo in the cell as well as the identity of

possible cargos remains to be resolved, there have been numerous reports of M10 being

implicated in various cellular processes.

Besides filopodia formation, other cellular processes in which M10 has been shown to play

a role include nuclear anchoring, spindle assembly, and axonal growth cone guidance (Weber et

al., 2004; Zhu et al., 2007; Toyoshima and Nishida, 2007). In 2004 it was demonstrated that the

MyTH4-FERM domain of M10 was able to bind directly to purified microtubules and disruption

of M10 function led to a disruption in nuclear anchoring, spindle assembly, and spindle-F-actin

association during meiosis (Weber et al., 2004). In a similar vein, in 2007, Toyoshima and

Nishida were further able to show that M10 was involved in properly orienting the mitotic

spindle parallel to the substratum in nonpolarized culture cells in a manner that was dependent

on integrin mediated adhesion. They were able to show that disruption of adhesion via pathways









that interfere with integrin-mediated adhesion caused misorientation of the mitotic spindle.

Furthermore, when they knocked out M10 using siRNA, they found that spindle orientation was

impaired much the same as it was when integrin-mediated adhesion was obstructed. In addition

to their findings that nonpolarized culture cells expressing normal M10 levels plated on culture

dishes coated with poly-L-lysine (a coating that precludes integrin-mediated adhesion) also

exhibited aberrations in mitotic spindle orientation, they were also able to successfully

demonstrate at least one other instance where M10 function was inhibited when upstream

integrin-mediated signaling was interrupted. In their work, Toyoshima and Nishida referred to

Weber et al.'s findings in 2004 that cited M10's ability to directly bind microtubules as a means

by which it could affect mitotic spindle orientation. Aside from its ability to interact with

microtubules, it has also been proposed that M10's PH2 domain may serve to properly target

M10 to the membrane in response to agonists that activate phosphatidylinositol 3-kinaase (PI3K)

by virtue the domain's ability to bind phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3]

(Tacon et al., 2004). However, this statement is in some opposition to Berg and Cheney's

observations that GFP-M10 constructs that were missing the head (motor) domain did not exhibit

proper localization to filopodial tips, but exhibited a diffuse localization pattern instead (Berg

and Cheney, 2002).

In addition to induction of filopodia, nuclear anchoring, and proper spindle formation, M10

has also been shown to play a role in nervous system development. Many other myosins have

also been previously demonstrated to play a role in nervous or sensory system development

owing to their roles as force-producing modulators of the actin cytoskeleton (Brown and

Bridgman, 2004). Specifically, in 2007, Zhu and colleagues published a report that

demonstrated M10's involvement in axonal path-finding. They found M10 plays an important









role in properly distributing netrin receptors in neuronal cells in order to correctly respond to

netrin-1 cues during the process of neurite outgrowth. This was demonstrated in vitro when

silencing of M10 prevented proper distribution of netrin receptors in neurites and in vivo by

showing that expression of a motor-less (non-functional) M10 was able to reduce neurite

outgrowth in response to netrin-1 in cortical explants derived from mouse embryos. This is one

of several instances in which it has been shown that MyTH4-FERM myosins play an important

role in maintenance of cellular projections in specialized cells (Oliver et al., 1999).









Table 1-1. List of S. flexneri TTSS effector proteins and their functions
Effector protein Function

IpaA Binds host cell vinculin to help depolymerize actin and mature
phagocytic cup during bacterial entry
IpaB Binds cell surface protein CD44 and associates with aP5i-integrin

Leads to induction of apoptosis in macrophages

Binds to IpaC to form Ipa complex that is embedded into host cell
membrane
IpaC Binds to IpaB to form Ipa complex that is embedded into host cell
Membrane

Triggers F-actin nucleation via C-terminal domain
IpaD Known to form polymers with IpaC

Known to be needed for binding and entry of /lgel//li
IpgD Specifically dephosphorylates PI(4,5)P2 to yield PI(5)P

Thought to aid in relaxing membrane-cytoskeleton interaction to
facilitate actin filament extensions during entry
VirA Triggers host microtubule destabilization and leads to membrane
ruffling during bacterial entry (Yoshida et al., 2002)









Table 1-2. List of cytoskeletal components found in mammalian cells
Cytoskeletal Role
component


Actin


Arp2/3 complex







Profilin




N-WASP


VASP

Rho family
proteins

ADF/cofilin

Capping proteins


Thymosin 0-4


47 kDa globular protein that can exist as monomers (G-actin) or
can be polarized into filaments (F-actin) in the cell.

Structural protein for the microfilament layer of mammalian cell
cytoskeleton
Complex comprised of actin-related protein ARP2, ARP3, and five
other subunits that is necessary for de novo nucleation of new
actin filaments in the cell.

Binds to the side of an existing actin filament and initiates growth of
a new filament at a 700 angle to the existing filament; leads to the
establishment of branched actin networks in the cell.
Binds G-actin monomers and ushers them to the barbed end of a
growing actin filament.

Enhances ATP-ADP exchange when new monomers are incorporated
into a growing actin filament.
Activates the Arp2/3 complex and acts as a scaffolding protein to
bring Arp2/3 and G-actin into spatial and functional proximity to
initiate the formation of actin filaments.
Simultaneously binds the growing F-actin filament and profilin-
actin complexes to aid in efficient filament elongation.
GTPases that, once activated in response to extracellular signals,
activate and recruit N-WASP to the cytosolic face of the plasma
membrane.
Bind to the sides of actin filaments and sever filaments to enhance
disassembly at the barbed end.
Bind to the barbed end of actin filaments to stop the growth of one or
more filaments so that others may be allowed enhanced growth to
propel the cell in the desired direction.
Sequesters actin monomers in order to maintain a cytoplasmic pool of
free actin monomers, which can then be used for rapid filament
elongation of F-actin (Dedova et al., 2006).









CHAPTER 2
METHODS AND MATERIALS

Cell Infections with S. flexneri and M10 Localization

Cell Cultures and Bacterial Strains

HeLa (human cervical cancer) cells, PtK2 (kangaroo rat kidney) cells, and Cos7 (African

green monkey kidney) cells were maintained at 370C and 5% CO2 in Dulbecco's Modified

Essential Media (DMEM) containing 10% fetal bovine serum and 5% penicillin/streptomycin

antibiotic solution (DMEM complete). S. flexneri strain 2457T, a virulent strain of serotype 2a

(Wei et al., 2003), was a kind gift from Dr. Marcia Goldberg, Massachusetts General Hospital.

Individual bacterial colonies were selected from a tryptic soy agar or brain heart infusion agar

plate containing 0.01% Congo Red dye to ensure bacterial virulence (Meitert et al., 1991).

Colonies were inoculated into tryptic soy broth (TSB) or brain heart infusion (BHI) and were

grown overnight in a shaker at 370C. The next day, a diluted culture was made from the

overnight culture and allowed to grow to an OD600 between 0.600 0.800 to ensure optimal

expression of the outer-membrane protein, IcsA (Gold berg et al., 1994; Stevens et al., 2006).

For experiments requiring Listeria monocytogenes, bacteria were inoculated into BHI and grown

overnight in a shaker at 370C.

Transfection with cDNA Plasmids

Transfections with various M10 cDNA plasmid constructs, an Akt-PH cDNA plasmid

construct, and a PLC6-PH cDNA plasmid (Figure 3-4) were accomplished using FuGene6

Transfection Reagent (Roche) or Lipofectin Reagent (Invitrogen) according to the

manufacturer's instructions. M10 plasmid constructs that were used were constructed as

described previously (Berg and Cheney, 2002; Bohil et al., 2006): full-length green fluorescent

protein-tagged (GFP)-M10, GFP-M10-HMM (includes head, neck, and proposed coiled-coil









domains), GFP-M10-AFERM (full-length M10 with the FERM domain deleted), and GFP-M10-

AMyTH4AFERM (full-length M10 with the MyTH4 and FERM domains deleted). The GFP-

Akt-PH plasmid (contains the PH domain of the Akt protein kinase, amino acids 1-167) and

GFP-PLC6-PH plasmid (contains the PH domain of PLC6, amino acids 1-170) was constructed

as described previously (Varnai and Balla, 1998). Cells were allowed to incubate at 370C and

5% CO2 overnight to allow for uptake and expression of cDNA.

Cell Invasion

S. flexneri invasion of semiconfluent cell monolayers was performed as described

previously with some modifications (Zeile et al., 1996). Briefly, bacteria in log phase were spun

down, washed, and resuspended in 1X PBS. Bacteria were then added to HeLa cells or Cos7

cells grown on 35-mm culture dishes at an MOI of 50 bacteria per cell and subsequently

centrifuged at 500x g for 15-30 minutes to allow for bacterial adhesion and entry. Culture dishes

were then incubated for another 30-90 minutes at 370C and 5% CO2 to allow for initiation of

bacterial actin-based motility and filopodia formation. Extracellular bacteria were then removed

by washing cells with IX PBS. Media containing gentamicin at a concentration of 10g/mL was

then added back to the culture dishes to prevent growth of extracellular bacteria and cells were

incubated at 370C and 5% CO2 for 10-15 min before being viewed via video microscopy. L.

monocytogenes invasion of semiconfluent HeLa cell monolayers was carried out as described

previously (Sidhu et al., 2005).

Immunofluorescence and Phalloidin Stain

For immunofluorescence experiments, HeLa cells infected with S. flexneri were treated

with 3.7% formaldehyde, then permeabilized with 0.2% Triton X-100. A blocking solution (5%

fetal bovine serum resuspended in IX PBS) was then added to cells, followed by primary

antibody at a concentration of 1:250 (tropomyosin monoclonal mouse antibody was purchased









from Sigma and M10 polyclonal rabbit antibody was a kind gift from Dr. Richard Cheney,

University of North Carolina at Chapel Hill). Lastly, a FITC-conjugated secondary antibody was

added. For experiments where F-actin filaments were stained with phalloidin (Molecular

Probes), phalloidin resuspended in blocking solution at a concentration of 1:400 was added to

cells following the permeabilization step.

LY294002 Treatment

For experiments using a PI3K inhibitor, LY294002 resuspended in serum free media (final

concentration 50[M) was added to .\/nge//al-infected cells one hour after initiation of infection

and left to further incubate at 370C and 5% CO2 for another 45-60 min to allow sufficient time

for formation of filopodia. At this point, cells were washed and media containing LY294002 and

gentamicin (10g/mL) was added back to cells. Cells were allowed to incubate at 370C and 5%

CO2 for an additional 10-15 min before filopodia were analyzed via video microscopy.

Video Microscopy

Time-lapse (live) and immunofluorescence (fixed) images were obtained using either a

Nikon (Tokyo, Japan) or Zeiss (Germany) inverted microscope connected to a cooled charge-

coupled device camera (Hamamatsu, model C5985). Images were analyzed using Metamorph

4.0 image software (Universal Imaging, West Chester, PA) or AxioVision Release 4.6 image

software (Carl Zeiss MicroImaging, Inc.).

Transfection with siRNA's

Transfections with control siRNA (purchased from either Qiagen or Dharmacon) and M10

siRNA (purchased from Qiagen as described previously by Zhang et al., 2004 or purchased from

Dharmacon as catalog number J-007217-06-0005) were accomplished using the RNAifect

Transfection Reagent (Qiagen) according to the manufacturer's instructions. Cells were allowed









to incubate at 370C and 5% CO2 for 48 h to allow for efficient knockdown of M10. A Western

blot was performed as described below to confirm knockdown of endogenous M10.

Western Blots

Western blots were performed using cytoplasmic extracts subjected to SDS-PAGE.

Confluent monolayers of either HeLa, Cos7, or Caco2 cells were washed once and subsequently

scraped in a 1501d volume of 1X PBS containing 10x protease inhibitor cocktail (Complete

Protease Inhibitor Cocktail tablets, Roche). The solution was then passage several times

through a 23- or 25-guage needle to lyse cells and shear DNA without causing proteolysis to

endogenous M10 levels. Samples were loaded onto a 7.5% polyacrylamide gel, then proteins

were transferred to a PVDF membrane (Millipore). A polyclonal M10 antibody raised in rabbit

was used at a dilution of 1:1000 to probe the membrane (M10 antibody was provided by Dr.

Richard Cheney, University of North Carolina at Chapel Hill) followed by an HRP-conjugated

secondary antibody at a dilution of 1:2000. Super Signal West Pico Chemiluminescent Substrate

was used for protein detection (Pierce). To verify equal protein loading, the membrane was

stripped using Restore Western Blot Stripping Buffer (Pierce) and re-blotted with monoclonal P-

actin antibody raised in mouse (Sigma) at a dilution of 1:5000 followed by an HRP-conjugated

secondary antibody at a dilution of 1:10000.

Plaque Assays

The plaque assay protocol was carried out as previously described (Oaks et al., 1985), with

some modifications. Briefly, HeLa cells were grown to confluency in 6-well plates containing

appropriate media at 370C and 5% CO2. Cells were then treated with either control siRNA or

M10 siRNA (Qiagen and Dharmacon) and allowed to incubate for 48 h at 370C and 5% CO2 to

allow for efficient knockdown of M10. On the day of infection, S. flexneri grown to log phase

were centrifuged, washed, and resuspended in IX PBS. Bacteria were added to cell monolayers









at an MOI of 5 bacteria per cell. Plates were then incubated at 370C and 5% CO2 for 90 minutes.

During this adsorption or attachment period, plates were rocked back and forth every 30 minutes

to ensure equal distribution of bacteria over cell monolayers. Next, an agarose overlay (2 mL per

well) consisting of appropriate media (serum-containing DMEM for HeLa cells or appropriate

serum-containing MEM for Caco2 cells), 10tg/mL of gentamicin, and 5% low-melting

temperature agarose (Fisher Scientific) was added to each well. Plates were left at room

temperature in a tissue culture hood for 10-15 minutes to allow for the agarose overlay to solidify

and then incubated overnight at 370C and 5% CO2. The next day, a secondary agarose layer (1

mL per well) consisting of appropriate media, 10[tg/mL of gentamicin, 5% low-melting

temperature agarose, and 0.1% neutral red dye (Sigma) to assist in visualization of plaques was

added to each well. Plates were then incubated at 370C and 5% CO2 for 24-48 hours and then

examined for the formation of plaques.









CHAPTER 3
RESULTS

M10 Localizes to S. flexneri Contained in Filopodia

Introduction

In eukaryotic cells, filopodium formation requires both actin polymerization and

reshaping of the plasma membrane. Since .\/lg//A,'s intracellular motility is dependent upon

actin polymerization, it seems plausible that one or more actin-plasma membrane linker proteins

may be involved in the process of filopodium formation that occurs subsequently. In relation to

our research interests, M10 stands out as a potential candidate in this facet of.\/ige//A,'s

pathogenicity because it has previously been shown to be an important factor in filopodium

formation in eukaryotic cells (Berg et al., 2002). It has also been shown that although actin

polymerization alone is necessary to drive various forms of eukaryotic cell motility, myosins can

oftentimes be implicated in powering certain processes along with actin (Berg et al., 2001).

Some such processes include signal transduction (Bahler, 2000) and establishment of polarity

(Yin et al., 2000). It is known that filopodium formation mediated by IcsA and subsequent

uptake by adjacent epithelial cells is an important facet for virulent .\/nie//t to establish a

successful infection in animals (Sansonetti et al., 1991). Since to date little is known about the

mechanisms) .\/nge//t undertakes to carry out this process, we sought to investigate whether

M10 played a role and, if so, whether its role could shed any light on the process as a whole.

Firstly, it was necessary to determine the presence or absence of M10 in ./ilge//a-laden filopodia.

We employed a GFP-M10 plasmid construct in order to visualize the location of wild type M10

in live cells during infection with S. flexneri and utilized immunofluorescence techniques to

determine the location of endogenous M10 in fixed cells that had been infected with S. flexneri.









M10 localizes to motile Shigella, but not motile Listeria in HeLa cells

A number of bacterial species such as S. flexneri, L. monocytogenes, and several

Rickettsia species are able to invade non-phagocytic host cells and subsequently usurp host cell

actin machinery and undergo actin-based motility (Gouin et al., 2005). While the end effect of

actin polymerization, namely a means of propulsion within the host cell, is the same for the

various bacterial species that utilize it, the mechanisms by which actin polymerization is initiated

varies from pathogen to pathogen. Specifically, S. flexneri activates the Arp2/3 complex to

initiate actin assembly indirectly by first binding and activating N-WASP, whereas L.

monocytogenes is able to directly bind and activate the Arp2/3 complex via ActA (Kocks et al.,

1992; Gouin et al., 2005). This difference in mechanisms for achieving the same goal of

initiating actin-based motility for both pathogens suggests that other differences may also exist in

latter parts of the motility process. By observing HeLa cells expressing a full-length GFP-M10

plasmid construct using video microscopy, we found that during infection M10 was localized

most notably along the sides of filopodia containing .\/nge/ll (figure 3-1A) and sometimes in

actin tails of motile .\1/ge//l Under the same experimental conditions we found that M10 failed

to localize to any Listeria-induced actin structures (figure 3-1B). To further verify these

findings, we performed immunofluorescence experiments on HeLa cells infected with S. flexneri.

These experiments confirmed that endogenous M10 was concentrated behind, as well as partially

alongside, bacteria that were found in filopodia and that some intracellular bacteria recruited

M10 at one pole (figure 3-2). These observations indicate that at least one host factor may be

involved in some aspects of motility and filopodium formation in S. flexneri pathogenesis, but

not in L. moncytogenes pathogenesis.









Tropomyosin localizes to Listeria-Induced, but not Shigella-Induced, Actin Tails

Previously, it had been noted that the actin-binding protein, tropomyosin, localized to

Listeria-induced actin structures (Dabiri et al., 1990). However, there are no documented reports

of whether or not tropomyosin is recruited to .\/nge//l-induced actin tails. Tropomyosin is able

to bind along the sides of polymerized actin filaments and, once bound, preclude the binding of

myosin heads to the actin filament (Cooper, 2002). We hypothesized that one reason why we

were observing M10 localization in .l/nge/h/-induced, but not Listeria-induced, actin tails was

because of the possibility that tropomyosin was absent from actin tails induced by .\/nge//i

Indeed, when \l/nge//, -infected HeLa cells were co-stained with phalloidin and a tropomyosin

antibody, there was no tropomyosin localization to bacteria-induced actin tails. This was in

contrast to Listeria-infected HeLa cells in which co-staining with phalloidin and a tropomyosin

antibody almost always revealed co-localization (figure 3-3). While additional factors and

processes may also be at play, we concluded that the presence of tropomyosin in Listeria-

induced actin tails, and its absence from .l/nge,// -induced actin tails, provides one clue as to why

M10 is recruited to the actin tails of the latter while being excluded from the former.

Tail Region of M10 Facilitates Shigella's Ability to Form Longer Filopodia

Introduction

Myosin-X is a 235 kDa protein that is characterized by a head (motor) domain that shares

<45% identity with other myosins, 3 IQ motifs, and a predicted coiled-coil region (suggests that

native M10 exists as a dimer in cells). The tail domain consists of a MyTH4 domain, a FERM

domain, and 3 PH domains (Berg et al., 2000). In 2002, Berg and Cheney discovered that M10

localizes to the tips of filopodia and undergoes forward and rearward motion within filopodia. In

order to target which regions) conferred the protein's ability to localize to the filopodial tips,

they generated several GFP plasmid constructs: GFP-M10, the full-length protein with a GFP









tag; GFP-M10-HMM, a heavy meromyosin (HMM)-like fragment which contains the head,

neck, and coiled coil regions; and GFP-M10-Tail, which constitutes the distal tail domain

including the PH domains, MyTH4 domain, and the FERM domain. Their findings revealed that

both the GFP-M10 and the GFP-M10-HMM constructs were able to localize to the tips of

filopodia in a pattern similar to that seen when using immunofluorescence to visualize

endogenous M10. They concluded, therefore, that the motor, or head, domain of M10 was the

region responsible for properly targeting M10 to filopodial tips and speculated that the various

domains within the tail region perhaps contribute to the transport of cargo of some kind. At this

point it remains unclear which cargo(s) may specifically be transported by the domains found in

M10's tail region, but several investigations have identified certain cellular binding partners for

these domains. For example, the second of the three PH domains has been shown to bind the

PI3K product, PI(3,4,5)P3 (Isakoff et al., 1998; Tacon et al., 2004). PI(3,4,5)P3 is known to

function as a second messenger molecule that acts to induce local actin polymerization (Insall

and Weiner, 2001). Furthermore, M10 was recently shown to be a downstream effector of PI3K

during phagocytosis in macrophages, demonstrating at least one functional role for M10 in cells

(Cox et al., 2002). Immediately following the last PH domain is a myosin tail homology 4

(MyTH4) domain. This domain is also found in class VII myosins, several unconventional

myosins (M4, M10, M12, and M15), and kinesin-like calmodulin binding protein (KLCBP),

which is a microtubule binding motor protein found in plants (Reddy et al., 1996; Oliver et al.,

1999). At this point in time, the MyTH4 domain has been shown to have the ability to bind

microtubules only in KLCBP (Narasimhulu and Reddy, 1998). Finally, the most C-terminal

domain found in M10 is the FERM domain. The domain is named for a group of cytoskeleton-

membrane linker proteins (band 4.1/ezrin/radixin/moesin) in which the domain was first









identified and attributed the function of having the ability to bind PI(4,5)P2, a molecule present

in the inner leaflet of the plasma membrane (Chishti et al., 1998; Tacon et al., 2004). Despite

this function among the traditional FERM proteins, the FERM domain found in M10 does not

bind PI(4,5)P2, but has been shown to bind P-integrins (Zhang et al., 2004). The ability of M10

to bind P-integrins has been shown to be important in cell processes such as integrin-dependent

adhesion and filopodial extension as well as neurite outgrowth and growth-cone guidance (Zhang

et al., 2004; Zhu et al., 2007).

Absence of M10 Tail Region Results in Shorter Shigella-Induced Filopodia

To determine which of the M10 tail region domains was important for .\ligel//A filopodia

length, we transfected Cos7 cells with one of several GFP-M10 constructs -- GFP-M10-AFERM,

GFP-M10-AMyTH4AFERM, and GFP-M10-HMM and compared .\/ngel/h -induced filopodia

lengths in these cells to control cells transfected with full-length GFP-M10. We opted to use

Cos7 cells because they contain a low background level of endogenous M10 (figure 3-5) and

they naturally do not express filopodia on their surface (Bohil et al., 2006). These conditions

would enable us to assess the effects of the exogenously expressed M10 constructs with minimal

interference from the native M10 population. Lengths of ./nge//At-induced filopodia in cells

transfected with GFP-M10-HMM were compared to cells transfected with GFP-M10. A 33%

decrease in average filopodial lengths was observed in GFP-M10-HMM transfected cells (figure

3-6). This observation led us to conjecture that the tail region of M10 was important for

.\l/ge//A,'s ability to form filopodia. This is not surprising, since the tail is the region where

various domains are found to which specific binding partners can bind and confer, in part,

diverse functionality among myosins (Krendel and Mooseker, 2005).









The MyTH4 and FERM Domains of M10 Are Not Required for Efficient Shigella-Induced
Filopodium Formation

Although little is known about which cellular components are required in the process of

filopodial formation for \lI.nge//, there have been some observations noted for both .\lnge// and

Listeria, another intracellular pathogen that usurps host cell actin and forms filopodia into

adjacent cells to disseminate during infection (Carlsson and Brown, 2006). In 2005, Pust and

colleagues reported that the host cell protein ezrin, a member of the family of proteins containing

a FERM domain, accumulated at the sites of Listeria-induced protrusions and were further able

to show that disruption of its ability to bind CD44, an integral membrane protein, hampered

Listeria's ability to efficiently form filopodia. For .\/ng//Iu, it has previously been shown that

the inhibition of myosin light chain kinase (MLCK) and, presumably, the inability of myosin II

to be phosphorylated leads to a marked decrease in cell to cell spread in Caco 2 cells (Rathman et

al., 2000). In both instances, it is the disruption of an involved host cell factor that is responsible

for the impairment of intercellular bacterial spread. Furthermore, in the case of.\/nge//A, the

contribution of a myosin is implied in the process. With this in mind, as well as the knowledge

that M10 contains a FERM domain, we proceeded to transfect Cos7 cells with either GFP-M10-

AFERM or GFP-M10-AMyTH4-AFERM and compare the resulting ./nllge//-induced filopodial

lengths to a control (Cos7 cells transfected with GFP-M10) to see if we could reproduce the

results from our experiment with GFP-M10-HMM and thereby identify the domain responsible

for facilitating proper filopodial length. We found that transfections with either GFP-M10-

AFERM or GFP-M10-AMyTH4AFERM did not yield /nlge/A -induced filopodia that were

shorter in length than those formed with full-length GFP-M10 transfected cells (figures 3-7A and

3-7B). These results led us to hypothesize that the required domain for proper filopodium

formation was the PH domain.









Inhibition of PI3K products and overexpression of GFP-Akt-PH Do Not Affect Shigella-
Induced Filopodial Lengths

PH domains are found in over 100 different cellular proteins and are characterized by

sequence similarity to two regions in pleckstrin, a major substrate of protein kinase C in platelets

(Kavran et al., 1998). Proteins containing PH domains are involved in various tasks in the cell,

including signaling processes, cytoskeleton organization, regulation of intracellular membrane

transport, and modification of membrane phospholipids. The types of proteins in which PH

domains are found are varied as well and can usually be sorted into groups based on

functionality. These groups of proteins include: Serine/Threonine protein kinases, Tyr protein

kinases, small G-protein regulators, endocytic GTPases, adaptors, phosphoinositide metabolizing

enzymes, and cytoskeletal associated proteins (Rebecchi and Scarlata, 1998). Two relatively

well-studied PH domains are that of PLC-61, a member of the phospholipase C family that

hydrolyzes PI(4,5)P2, and that of Akt, a proto-oncogenic ser/thr kinase. While the PH domain of

PLC-61 specifically binds PI(4,5)P2 (Pawelczyk and Lowenstein, 1993), the PH domain of Akt

preferably binds phosphatidylinositol products of PI-3 kinase (PI3K) such as PI(3,4,5)P3 and

PI(3,4)P2 (James et al., 1996; Franke et al., 1997). Binding of PH domains in these and other

proteins to various phosphatidylinositol phospholipids in the plasma membrane and intracellular

membranes is usually for the purpose of regulating protein activity and/or targeting the protein to

its required intracellular location (Lemmon and Ferguson, 2001; Tacon et al., 2004).

Since neither the GFP-M10-AFERM nor the GFP-M10-AMyTH4AFERM constructs had

any effect on .\/n gel/h-induced filopodial lengths even though lengths were shorter when the

GFP-M10-HMM construct was used, we hypothesized that one or more of the PH domains in the

M10 tail region was responsible for the role M10 was playing in enabling \/ngel//t to efficiently

form filopodia. It has previously been shown that the M10 PH2 domain binds PI(3,4,5)P3









(Tacon et al., 2004). With this in mind, we sought to abrogate this domain's functionality using

two approaches first, we chose to specifically inhibit PI3K, by whose activity PI(3,4,5)P3 is a

product, by using the reversible inhibitor LY294002 (Vlahos et al., 1994) and second, we

overexpressed the PH domain of Akt, which is known to bind PI(3,4,5)P3, as a way to compete

out the native binding partner for the M10 PH2 domain. We wanted to use two approaches since

we weren't sure whether the binding partner was a host cell factor or a bacterial component. We

speculated that if the binding partner was a host cell factor, then both approaches would result in

filopodia of shorter length similar to the results we obtained when we transfected cells with GFP-

M10-HMM. However, if it was a bacterial component, then inhibiting PI3K activity might not

have any effect on filopodial length while overexpression of GFP-Akt-PH should compete out

the native binding partner no matter where it originated. Surprisingly, when HeLa cells infected

with ./nge//At were either transfected with GFP-Akt-PH or treated with LY294002, there was no

significant change in .\Vllge/a-induced filopodial lengths observed (figures 3-8A 3-8C). To

rule out the involvement of other phosphatidyl phosphoinositide groups, we also repeated these

experiments in HeLa cells transfected with GFP-PLC6-PH. In these experiments, filopodia

formed from cells transfected with GFP-PLC6-PH did not differ in length from filopodia formed

from cells transfected with GFP only (figure 3-8C). These findings seem to indicate that there

may either be redundant factors that can compensate for the abrogation of M10's PH2 domain's

functionality or that the presence of one of the other PH domains within the M10 tail region is

able to mediate the functionality required for ,'\/Nge//Al to form filopodia.









Reduction of Endogenous M10 Levels Curtails S. flexneri Cell-to-Cell Spread in HeLa Cell
Monolayers In Vitro

In order to establish a successful infection in the host, it is necessary for \/nhge//t to be able

to move from an infected cell to neighboring cells along the colonic epithelium (Sansonetti,

2001). This process of intercellular advancement by ./nge,//At causes the formation of

hemorrhagic plaques within the colonic epithelium in the infected host. There are a few

experimental procedures that have been devised to test ,.s/ge//Al's virulence capacity in this

aspect. Two such tests include the Sereny test (keratoconjunctivitis shigellosa), in which virulent

bacteria are placed on the conjunctiva of the guinea pig eye resulting in a rapid spread of

keratoconjunctivitis (Sereny, 1955) and a cultured cell monolayer plaque assay (Oaks et al.,

1985), in which virulent bacteria are allowed to infect a confluent monolayer of cultured HeLa

cells. The plaque assay requires an agarose overlay on the cultured cell monolayer so that the

formation of plaques can be observed after a few days post-infection. It is known that virulent S.

flexneri strains invade HeLa cells with high efficiency and those strains that are capable of

undergoing cell-to-cell spread in cultured cell monolayers often lyse or otherwise kill the

infected cells leaving behind a zone of clearing (i.e., plaques) in the monolayer (Oaks et al.,

1985; Sansonetti et al., 1986).

In order to further assess M1O's importance in ,\/lge//' 's ability to efficiently form

filopodia and thereby carry out efficient intercellular spread, we decided to test what effect

knocking down endogenous M10 levels using siRNA would have on ,\s/lge//l's ability to form

plaques on a cultured HeLa cell monolayer. When compared to HeLa cell monolayers treated

with a non-targeting control siRNA, .\/ige/ll were less able to form plaques in HeLa cell

monolayers treated with M10 siRNA to knock down endogenous M10 levels. We found there

were 26% fewer plaques formed in M10 siRNA-treated HeLa cell monolayers compared to









control siRNA-treated monolayers (figure 3-9). These observations are consistent with our

previous observation that .\/ng//al-induced filopodial lengths measured in HeLa cells transfected

with M10 siRNA were 30% shorter, on average, compared to those measured in HeLa cells

transfected with ctrl siRNA (Figure 3-10).


Figure 3-1A. Time-lapse pictures of GFP-M10 localizing to motile intracellular Shigella in
PtK2 cells. Pictures were taken at 20s intervals. Arrows indicate nodules of
moving GFP-M10 alongside motile Shigella.









































Figure 3-1B.


Time lapse pictures of PtK2 cells transfected with GFP-M10 and infected with
Listeria. GFP-M10 does not localize to motile intracellular Listeria. Arrows
indicate motile intracellular Listeria. Arrowheads indicate normal localization of
GFP-M10 in host cell filopodial tips.











Vr eel
.. .. _. *.
,
-. d. ^i
v^ ^C vy
^^-<**- ^'
tt ^^^


M10 antibody localizes to intracellular Shigella and Shigella-laden filopodia. A,
C, and E are phase images. B, D, and F are corresponding immunofluorescence
images.


Figure 3-2.
















































Tropomyosin monoclonal antibody localizes to Listeria-, but not Shigella-induced
actin tails in HeLa cells. A and B depict Listeria-infected cells; C and D depict
Shigella-infected cells (green FITC phalloidin; red tropomyosin monoclonal
antibody).


Figure 3-3.








GFP-Akt-PH



GFP-PLC-PH


MIO Tail


GFP-M10


GFP


GFP-M10-HMM




GFP-M1O-xFERM


M10 1M10
ed coi8ed
**H~fB coil


M10
coiled
coil


MIO Tail


M10
coiled -
coil


MIO Tail


GFP-MO1-xMyTH4
xFERM


The GFP-tagged cDNA constructs used in cell transfections.


H0M10
coiled
H a coil


Figure 3-4.


GFP


--GFP


GFP




GFP
IGF- ~


GFP


PL-P












Cos7
Cos7 extract +
extract GFP-M10


250 kDa

75 kDa


Figure 3-5.








20 -




15 -




S10-




5-




0 -
Figure 3-6.


Western blot showing endogenous M10 levels in Cos7 cell extracts. Lane labeled
"Cos7 extract" represents protein content of non-transfected Cos7 cells. Lane
labeled "Cos7 extract + GFP-M10" represents protein content of Cos7 cells
transfected with GFP-M10. 75kDa band represents an unknown protein with
which the M10 antibody cross-reacts and serves as a loading control.


H GFP-M10

] GFP-M1O-HMM


Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-HMM,
infected with Shigella, and analyzed using video microscopy. The average
filopodial lengths are represented. On average, .\l/gel// formed filopodia that
were 33% shorter in Cos7 cells transfected with GFP-M10-HMM compared to
Cos7 cells transfected with GFP-M10 (11.66[tm compared to 15.25utm,
respectively. P value is 0.006). For GFP-M10 (black bar) and GFP-M10-HMM
(gray bar), n = 24 and 44, respectively. Data is representative of three
independent experiments.


M10 Ab


















T



10



0



0 -




Figure 3-7A. Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-
AFERM, infected with Shigella, and analyzed using video microscopy. The
average filopodial lengths are represented. There was no significant difference
when Shigella-induced filopodial lengths from Cos7 cells transfected with GFP-
M10-AFERM were compared with those from Cos7 cells transfected with GFP-
M10 (11.1O0m compared to 12.22pm, respectively. P value is 0.54). For GFP-
M10 (black bar) and GFP-M10-AFERM (gray bar), n = 21 and 27, respectively.
Data is representative of three independent experiments.























f GFP-MO T

] GFP-MI0-xI'yTH4xFERM


Figure 3-7B.


Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-
AMyTH4AFERM, infected with Shigella, and analyzed using video microscopy.
The average filopodial lengths are represented. There was no significant
difference when .\/nlge// -induced filopodial lengths from Cos7 cells transfected
with GFP-M10-AmyTH4AFERM were compared with those from Cos7 cells
transfected with GFP-M10 (12.76km compared to 12.77gm, respectively. P value
is 0.65). For GFP-M10 (black bar) and GFP-M10-AFERM (gray bar), n = 36 and
26, respectively. Data is representative of three independent experiments.


10 -






*r 5-


0 ----L


















15- T


[] DIAO (CtA)

10- [] LY294002




5-




0 -
Figure 3-8A. HeLa cells were infected with Shigella, treated with either DMSO (control) or LY
294002, and analyzed using video microscopy. The average filopodial lengths are
represented. There was no significant difference when ./w ge//l -induced
filopodial lengths from DMSO-treated HeLa cells were compared with
LY294002-treated HeLa cells (13.88tm compared with 15.00tm, respectively. P
value is 0.49). For DMSO-treated cells (black bar) and LY 294002-treated cells
(gray bar), n = 45 and 54, respectively. Data is representative of three
independent experiments.



















" 10-





- -


Figure 3-8B.


GFP-Akt-PH

Ctri


HeLa cells were either transfected with GFP-Akt-PH or not transfected (control),
infected with Shigella, and analyzed using video microscopy. The average
filopodial lengths are represented. There was no significant difference when
\/nlg//al-induced filopodial lengths from HeLa cells transfected with GFP-Akt-PH
were compared with those from non-transfected HeLa cells (12.30pm compared
with 12.27upm, respectively. P value is 0.97). For cells transfected with GFP-Akt-
PH (black bar) and non-transfected cells (gray bar), n = 26 and 50, respectively.
Data is representative of three independent experiments.























] GFP nly(Ctrl)

C GFP-PLC-PH


Figure 3-8C.


HeLa cells were either transfected with GFP-PLC6-PH or GFP only, infected with
./Nhge//i, and analyzed using video microscopy. The average filopodial lengths
are represented. There was no significant difference when .'/Nlge//a-induced
filopodial lengths from HeLa cells transfected with GFP-PLC6-PH were
compared with those transfected with GFP only (16.12ipm compared with
15.54pm, respectively. P value is 0.59). For cells transfected with GFP (black
bar) and GFP-PLC6-PH (gray bar), n = 43 and 36, respectively. Data is
representative of three independent experiments.


15-


B
*i-

10-


5-










Cttr M1O
sIRNA sIRNA


250 kDa


40 kDa


100 -



75 -


MO1 Ab


ActinAb













E CtrlsiRNA
SMIOsiRNA


Bar graph showing number of plaques formed on HeLa cell monolayers by S.
flexneri. HeLa cells were treated with either control siRNA or M10 siRNA.
Western blotting revealed -80% reduction of endogenous M10 in HeLa cells
treated with M10 siRNA. "Control" and "M10 siRNA"-treated HeLa cell
monolayers were infected with S. flexneri and subsequent plaque formation was
enumerated. The numbers of plaques counted for Control cells and M10 siRNA
cells was 92 and 68, respectively. P value is 0.0803.


Figure 3-9.


0 ---























M Ctrl siRNA

I MI0 siRNA


Figure 3-10.


HeLa cells were either transfected with non-targeting siRNA (Ctrl siRNA) or
M10 siRNA, infected with \/igel//li, and analyzed using video microscopy. The
average filopodial lengths are represented. There was significant difference when
.'/ilge//'l-induced filopodial lengths from HeLa cells transfected with Ctrl siRNA
were compared with those transfected with M10 siRNA (14.73 km compared with
11.01[ m, respectively. P value is 0.0005). For cells transfected with Ctrl siRNA
(black bar) and M10 siRNA (gray bar), n = 32 and 35, respectively. Data is
representative of three independent experiments.


15 -




S10-



[M.
5-


0 -1









CHAPTER 4
DISCUSSION

M10 Recruitment to Shigellaflexneri

An important facet of pathogenesis for any pathogen is the ability to establish an

infectious foothold in the host to propagate the infection. .\l/ge//Jflexneri shares similarities

with other intracellular pathogenic bacteria such as species of Listeria, Rickettsia,

Mycobacterium, and Burkholderia in terms of the mechanism of actin-based intra- and

intercellular motility it has adopted to spread an infection in the host (Carlsson and Brown,

2006). Due to the crucial role intercellular spread plays in successfully establishing infection, it

is important to have a better understanding of the factors and mechanisms that govern ,/nge/a t-

induced filopodium formation. We have successfully shown that an unconventional myosin,

M10, is recruited to intracellular motile ,.lghl/ht undergoing filopodia formation and that its

presence seems to promote the efficient formation of filopodia. M10 has previously been

implicated in the process of filopodia formation in eukaryotic cells (Berg and Cheney, 2002;

Bohil et al., 2006) and this is the first time that this myosin has been implicated in the efficient

formation of filopodia in an intracellular bacterium. By either reducing endogenous levels of

M10 in cells or expressing GFP-M10-HMM, we have shown that ./nge//lt are less able to form

filopodia of normal length when compared to unaltered cells (Figure 3-6 and 3-10). The inability

of bacteria to form filopodia of normal length also seems to lead to a slowing down in the

process of cell-to-cell spread as is evidenced by the fewer number of plaques formed by .,/nge/A/t

on HeLa cell monolayers that have been treated with M10 siRNA compared to those formed on

monolayers treated with control siRNA (Figure 3-9).

Since cells expressing GFP-M10-HMM yielded .l/nlge/l -induced filopodia of lengths

similar to cells in which endogenous M10 had been knocked down, we supposed that the ability









of M10 to aid in bacterial filopodia formation was due to one or more domains within the tail

region. We found it difficult, however, to pinpoint the domain(s) that contained this property.

Although Bohil and colleagues (2006) were able to show that deletion of the MyTH4-FERM

region within the M10 tail was sufficient to nullify M10's ability to induce filopodia formation in

eukaryotic cells, we found no indication that this was the case for .l/gell, /-induced filopodia

(Figure 3-7A and 3-7B). Furthermore, when we tried to inhibit the PH2 domain within the M10

tail region, we found no reduction in .\/lgel/h-induced filopodia lengths as compared to cells

expressing the GFP-M10-HMM (Figure 3-8A C). Our results show that, in ./lNge//al

pathogenesis, only the complete absence of the entire M10 molecule or simply the absence of the

tail region hinders its ability to efficiently form filopodia and undergo efficient cell-to-cell

spread. This seems to indicate that .\/ige/ll is not relying on the presence of any cargo that the

M10 tail region might be delivering in order to form filopodia, but rather requires the presence of

the tail region perhaps to assist in proper localization and/or functionality of M10. It is

interesting to note that in a recently published paper, Tokuo and colleagues (2007) demonstrated

that only the motor function of the two-headed form of M10 is crucial in initiating filopodia

formation in eukaryotic cells, whereas, in regards to the process of.//ige//t pathogenesis, our

findings seem to indicate that the presence of the PH domain-containing tail region is also

required. It should be noted, however, that the filopodia observed in the Tokuo et al. study were

only borne of preexisting lamellipodia in migrating cells and were relatively transient, quickly

being retracted back into the lamellipodia. This is in some opposition to the study reported by

Bohil et al. where they showed that the ability of M10 to induce filopodia formation was

abolished when the MyTH4-FERM domains were deleted from the tail region.









M10 Contribution During Shigella-induced Filopodium Formation

In this study we have relayed one of the first reports of an unconventional myosin being

involved in efficient .\/lge/ll-induced filopodia formation and cell-to-cell spread. It has

previously been reported that myosin motors are not involved nor required in the process of

actin-based motility carried out by S. flexneri (Loisel et al., 1999). Furthermore, there have been

previous reports of myosins being recruited to the phagocytic cup formed by .l/nge/lt upon entry

into nonphagocytic host cells (Clerc and Sansonetti, 1987; Graf et al., 2000), but their possible

involvement in the process of filopodial formation has not been intensively investigated. Some

evidence pointing to the involvement of host cell myosins in .\ligel//a cell-to-cell spread was

reported in 2000 by Rathman and colleagues. In their work, they found that the inhibition of

MLCK, which is known to phosphorylate the light chain of myosin II, resulted in a marked

decrease in .\/lge/la's ability to disseminate in cultured Caco2 cell monolayers. Our study

demonstrates that an unconventional myosin motor protein, M10, is recruited to motile

intracellular .l/nge/lt as well as ./nge//At bound in filopodia (figure 3-2). We were also able to

show that M10 recruitment was specific to .\l/ge/ll and was not recruited to another intracellular

pathogen that employs actin-based motility, L. monocytogenes (figure 3-1B). There is a

similarly intriguing finding reported by Kolesnikova et al. (2006) of M10's co-involvement with

actin in the budding release of a particular virus. This group observed that the budding release of

Marburgvirus (MARV) particles occurred almost exclusively at filopodia. When polymerization

of actin in filopodia was inhibited, this resulted in a marked decrease in total virus particles being

released into the extracellular medium. Moreover, when M10 and/or Cdc42 were inhibited, the

intracellular localization of a matrix protein known to play a key role in the release of MARV

particles was concomitantly inhibited. In lieu of these findings the group went on to further

theorize that perhaps by preferentially utilizing host cell filopodia at egress sites, the virus was









capitalizing on these cellular projections' close contact with neighboring cells and thereby

enhancing the chances of successful invasion of adjacent cells. To date, this is the first finding

of its kind to be reported in the field of viral pathogenesis and creates a striking parallel to our

findings with M10 and S. flexneri's means of intercellular spread.

At this point it isn't yet clear whether the recruitment of M10 to .\l/ge//At is mediated by a

host cell factor or a bacterial component. There have only been a few studies examining

,s\/ge/Al,'s involvement in mediating efficient cell-to-cell spread. One recent report cites the

ability of the TTSS-secreted effector protein VirA to sever microtubule filaments as an important

means of facilitating optimal intra- and intercellular spread (Yoshida et al., 2006).

Involvement of M10's Tail Region in Shigella-Induced Filopodium Formation

The results we obtained from our experiment using Cos7 cells transfected with GFP-M10-

HMM that were then infected with ./nge//At indicated that the tail region of M10 contributes to

some extent in aiding i.\/gel/,-induced filopodium formation (figure 3-6). For many myosins,

the tail region is usually the area in which domains are found that confer cargo-binding

specificity (Sellers, 2000). In the case of M10, many findings have been reported that cite one

or more of its tail domains responsible for conferring some functionality to M10 in a broad array

of cellular functions. Although no one specific function has been attributed to M10, it is mostly

known for its role as a potent inducer of filopodia on the surface of mammalian cells (Berg and

Cheney, 2002; Bohil et al., 2006). In 2002, Berg and Cheney were able to show that the head

(motor) domain was responsible for properly localizing M10 to filopodial tips while Bohil and

colleagues were later able to show that the MyTH4-FERM region was responsible for M1O's

ability to promote filopodia formation (Bohil et al., 2006).









Future Directions

One of the main questions remaining about .,/Nge//at's ability to recruit host cell M10 is

whether the recruitment is due to the presence of a bacterial or host cell factor. One possibility is

that the absence of tropomyosin in .\/igie//a-induced actin tails (Figure 3-3) simply makes actin

available for M10 binding. However, it is unlikely that this would be the sole reason if.'/ge//at

is in fact deliberately recruiting M10 for a specific task. If.\'l/ge//At were recruiting M10 by way

of a bacterial factor, one candidate could be the IcsA protein. This protein stands out because its

polarized distribution at one pole of motile bacteria is what enables directional motility in

,\l//ge/At (Goldberg and Theriot, 1995) and because its reported distribution pattern closely

resembles the pattern we see in M10 localization. Furthermore, this would be consistent with

our finding that M10 recruitment is specific to .\l/ge//At and not Listeria, another intracellular

pathogen that utilizes actin-based motility via expression of the surface protein ActA (Gouin et

al., 2005).

Alternatively, if M10 were recruited to intracellular ./nge//At due to binding of a host cell

factor, the absence of tropomyosin in actin tails could facilitate M10 motor activity while

binding of host cell factors by the tail region could facilitate proper localization of the myosin

molecule. This idea has been proposed previously (Tacon et al., 2004) and it is thought that the

three tandem PH domains found in the M10 tail region are likely to be candidates for proper

intracellular localization of M10. If this were the case, it may explain why we see truncated

filopodia in cells expressing a M10-HMM construct, but not in cells expressing a M10-AFERM

or a M10-AMyTH4-AFERM construct. The reason why filopodia lengths are not truncated in the

presence of compounds that hinder PH domain activity, however, remains elusive. It may be

possible that a sequence or set of sequences not associated with a defined domain exists in the

region between the coiled-coil domain and the MyTH4 domain. It could be possible that the









presence of sequences in this region facilitate binding of the tail region to other proteins that

could play a role in proper intracellular M10 localization.









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Bahler, M. 2000. Are class III and class IX myosins motorized signaling molecules? Biochem.
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Bear, J.E., T.M. Svitkina, M. Krause, D.A. Schafer, J.J. Loureiro, G.A. Strasser, I.V. Maly, O.Y.
Chaga, J.A. Cooper, G.G. Borisy, and F.B. Gertler. 2002. Antagonism between
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Bennish, M.L. and B.J. Wojtyniak. 1991. Mortality due to shigellosis: community and hospital
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Berg, J.S., B.H. Derfler, C.M. Pennisi, D.P. Corey, and R.E. Cheney. 2000. Myosin-X, a novel
myosin with pleckstrin homology domains, associates with regions of dynamic actin. J.
Cell. Sci. 19:3439-3451.

Berg, J.S., B.C. Powell, and R.E. Cheney. 2001. A millennial myosin census. Mol. Biol. Cell.
12:180-794.

Berg, J.S. and R.E. Cheney. 2002. Myosin-X is an unconventional myosin that undergoes
intrafilopodial motility. Nat. Cell. Biol. 4:246-250.

Bemardini, M.L., J. Mounier, H. d'Hauteville, M. Coquis-Rondon, and P.J. Sansonetti. 1989.
Identification of icsA, a plasmid locus of .\l/gel/l flexneri that governs bacterial intra-
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BIOGRAPHICAL SKETCH

Ellen Antoun Bishai was born on June 14, 1980 in Edmonton, Alberta, Canada. She and

her family moved to Jersey City, NJ in 1983 and finally settled in Tampa, FL in 1991. Ellen

completed her undergraduate studies at the University of South Florida in Tampa where she

majored in biology and was an active member in the viola section of the University Orchestra.

Upon graduating in the Spring semester of 2003, she was enrolled in the Interdisciplinary

Program in Biomedical Sciences (IDP) at the University of Florida in the Fall semester of that

year. There, she worked under the guidance of Dr. Frederick Southwick in the completion of

this dissertation. Ellen intends to continue her education by attending the University of

Pennsylvania, the University of Georgia, or the University of Florida Veterinary School in

pursuit of a doctor of veterinary medicine degree.





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1 Shigella flexneri RECRUITS HOST CELL MYOSIN-X FOR EFFICIENT FORMATION OF FILOPODIA By ELLEN A BISHAI 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

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2 2007 Ellen A Bishai

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3 To God, without whose hand this would not have been possible; and to my family whose love and prayers have always provide d a fervent base of support.

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4 ACKNOWLEDGMENTS As I already stated, I would like to thank my family for all their love, prayers, and support throughout my life and these la st few years. I am also greatly indebted to my mentor who was always a wonderful source of encourag ement and motivation. I honestly wouldnt have made it through this experience without his co ntinual guidance and s upport. Lastly, but definitely not least, I would like to thank all of my lab-mates, past and present. Not only did they provide me with technical guidance when it wa s needed, but their frie ndship was a valuable resource in and of itself.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 Significance and Epidemiology..............................................................................................11 Filopodium Formation in Eukaryotic Cells............................................................................18 Myosins and Filopodia Formation..........................................................................................20 Overview of Myosins......................................................................................................20 Myosin-X (M10) and Filopodia Formation.....................................................................21 2 METHODS AND MATERIALS...........................................................................................27 Cell Infections with S. flexneri and M10 Localization...........................................................27 Cell Cultures and B acterial Strains..................................................................................27 Transfection with cDNA Plasmids..................................................................................27 Cell Invasion.................................................................................................................. ..28 Immunofluorescence and Phalloidin Stain......................................................................28 LY294002 Treatment......................................................................................................29 Video Microscopy...........................................................................................................29 Transfection with siRNAs.....................................................................................................29 Western Blots.................................................................................................................. ........30 Plaque Assays.................................................................................................................. .......30 3 RESULTS........................................................................................................................ .......32 M10 Localizes to S. flexneri Contained in Filopodia.............................................................32 Introduction................................................................................................................... ..32 M10 localizes to motile Shigella but not motile Listeria in HeLa cells.........................33 Tropomyosin localizes to Listeria -Induced, but not Shigella -Induced, Actin Tails.......34 Tail Region of M10 Facilitates Shigella s Ability to Form Longer Filopodia.......................34 Introduction................................................................................................................... ..........34 Absence of M10 Tail Region Results in Shorter Shigella -Induced Filopodia................36 The MyTH4 and FERM Domains of M10 Are Not Required for Efficient Shigella Induced Filopodium Formation...................................................................................37 Inhibition of PI3K products and overexpr ession of GFP-Akt-PH Do Not Affect Shigella -Induced Filopodial Lengths...........................................................................38

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6 Reduction of Endogenous M10 Levels Curtails S. flexneri Cell-to-Cell Spread in HeLa Cell Monolayers In Vitro ....................................................................................................40 4 DISCUSSION..................................................................................................................... ....54 M10 Recruitment to Shigella flexneri .....................................................................................54 M10 Contribution During Shigella -induced Filopodium Formation......................................56 Involvement of M10s Tail Region in Shigella -Induced Filopodium Formation...................57 Future Directions.............................................................................................................. ......58 LIST OF REFERENCES............................................................................................................. ..60 BIOGRAPHICAL SKETCH.........................................................................................................69

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7 LIST OF TABLES Table page 1-1 List of S. flexneri TTSS effect or proteins and their functions...........................................25 1-2 List of cytoskeletal components found in mammalian cells..............................................26

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8 LIST OF FIGURES Figure page 3-1A Time-lapse pictures of GFP-M10 localizing to motile intracellular Shigella in PtK2 cells ......................................................................................................................... ...41 3-1B Time lapse pictures of PtK2 cells tran sfected with GFP-M10 and infected with Listeria ...................................................................................................................... ........42 3-2 M10 antibody localizes to in tracellular Shigella and Sh igella-laden filopodia.................43 3-3 Tropomyosin monoclonal antibody localizes to Listeria-, but not Shigella-induced actin tails in HeLa cells......................................................................................................44 3-4 The GFP-tagged cDNA constructs used in cell transfections...........................................45 3-6 Cos7 cells were transfected with eith er full-length GFP-M10 or GFP-M10-HMM, infected with Shigella, and an alyzed using video microscopy..........................................46 3-7A Cos7 cells were transfected with ei ther full-length GFP-M10 or GFP-M10FERM, infected with Shigella, and an alyzed using video microscopy..........................................47 3-7B Cos7 cells were transfected with ei ther full-length GFP-M10 or GFP-M10MyTH4 FERM, infected with Shigella, and analyzed using video microscopy...........48 3-8A HeLa cells were infected with Shigella, tr eated with either DM SO (control) or LY 294002, and analyzed using video microscopy.................................................................49 3-8B HeLa cells were either transfected with GFP-Akt-PH or not transfected (control), infected with Shigella, and an alyzed using video microscopy..........................................50 3-8C HeLa cells were either transfected with GFP-PLC -PH or GFP only, infected with Shigella and analyzed using video microscopy................................................................51 3-9 Bar graph showing number of plaque s formed on HeLa cell monolayers by S. flexneri ..............................................................................................................................52 3-10 HeLa cells were either transfected with non-targeting siRNA (Ctrl siRNA) or M10 siRNA, infected with Shigella and analyzed using video microscopy.............................53

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9 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 Shigella flexneri RECRUITS HOST CELL MYOSIN-X FO R EFFICIENT FORMATION OF FILOPODIA By Ellen Bishai December 2007 Chair: Frederick S. Southwick Major: Medical Sciences Immunology and Microbiology Shigella flexneri is a gram negative rod that causes Shigellosis, a highly communicable disease responsible for significant morbidity and mortality in underdeveloped countries. Shigella is able to enter the cytoplasm of gastrointestinal epithelial cells and subsequently usurp host cell actin to move intracellularly and to the cell periphery to form finge r-like filopodia. These protrusions are ingested by neighboring cells, allowing Shigella to spread from cell to cell and pr oduce hemorrhagic plaques. Much work has been done to delineate the factors and mechanisms necessary for Shigella -induced actin-based intracellular motility. In contrast, the factors and mechanisms underlying filopodia formation by this bacterium are not well characterized. The unconventiona l myosin, myosin X, has recently been shown to contribute to filopodia formation in various mammalia n cell lines (Berg, 2002). We found that in living HeLa cells, GFP-Myosin X concentrates along the sides of filopodia containing Shigella Immunofluorescence microscopy utilizing a specific an ti-myosin X antibody corroborated these findings. Furthermore, Listeria monocytogenes another intracellular pathogen th at usurps host cell actin for motility, fails to recruit GFP-Myosin X. K nocking down endogenous myosin X levels in Shigella infected cells using myosin X-specific siRNA results in approximately a 30% reduction in Shigella induced filopodia formation when compared to cells containing native levels of myosin X (avg 11.01 m compared to 14.73 m, respectively; p=0.0005). Additionally, transfection of living HeLa cells with a GFP-Myosin X-HMM construct (contains only head, neck and coiled coil domains of myosin X) resulted

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10 in a significant reduction in the lengths of Shigella -induced filopodia when comp ared to cells transfected with full-length GFP-Myosin X (avg 11.66m compar ed to 15.25m, p = 0.006). We conclude that myosin X is an important component in filopodia formation by Shigella flexneri and that the tail region is responsible for conveying this function.

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11 CHAPTER 1 INTRODUCTION Significance and Epidemiology Shigellosis, also known as acute bacillary dysentery, is caused by Shigella spp. and annually affects 164 million people worldwide and cau ses 1.1 million deaths w ith the majority of cases occurring in children livi ng in developing countries (Je nnison and Verma, 2004). The disease is characterized by loose stools contai ning blood and pus as well as fever, abdominal cramps, and tenesmus (an incomplete sens e of evacuation with rectal pain) (Sur et al. 2004). The disease is highly communicable and is spread via the feca l-oral route, which can be facilitated by poor water sani tation, poor hygiene, and close personal contact. The higher incidence of disease spread in developing countries is often attributed to these conditions (Jennison and Verma, 2004). Countries where epidemics have been reported include South American countries, Asian countries ( i.e. Bangladesh, Sri Lanka, Maldives, Nepal, Bhutan, and Myanmar), and regions in southern and eastern India (Sur et al. 2004). Although the disease primarily affects populations living in developing countries, travelers visiting endemic areas may also be infected if they do not take proper precautionary hygienic measures (Sur et al ., 2004). Additionally, in the United States outbreaks that originate in day care centers are not uncommon and can subsequently spread throughout communities (Shane et al ., 2003). The etiological agents of Sh igellosis belong to the genus Shigella and include four species: S. dysenteriae (16 serotypes, of which serotype 1 is the most deadly due to its ability to produce shiga toxin), S. flexneri (6 serotypes, most of which are responsible for endemic disease in developing countries with seroty pe 2a being the most prevalent), S. boydii (8 serotypes) and S. sonnei (1 serotype, commonly the source of day care center outbreaks) (San sonetti 2001). Being

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12 endemic in most developing countries, S. flexneri has been reported to cause more mortality than any other Shigella species (Bennish a nd Wojtyniak, 1991; Kotloff et al. 1999). Pathogenesis of S. flexneri Shigella flexneri is a gram-negative, nonsporulating, facultative anaerobic rod that causes an invasive infection of the human colon. The key factors involved for successful pathogenesis of S. flexneri include 1) the ability to traverse the co lonic epithelium; 2) the ability to induce uptake into the non-phagocytic cells of the colonic epithelium; and 3) the ability to usurp the host cells actin machinery in order to facilitate intr aand intercellular motility, which result in the spreading of the infection (Sansonetti 2001). Colonic Epithelium: Invasion and Entry Once ingested, S. flexneri is able to pass through the st omach unharmed. This attribute enables S. flexneri to successfully establish an inf ection even if only a small number of microorganisms is ingested (as few as 10-100 microorganisms) (Sansonetti 2001). The reason why only a small number of microorganisms is suffi cient to establish an infection is believed to be the presence of acid-resistance pathways possessed by the bacteria that are induced upon encountering hostile acidic environments such as the human stomach (Gianella et al ., 1972; Jennison and Verma, 2007). The two pathways thought to play a role in Shigella acid-resistance are the acid-resistance pathway 1 (AR1) a st ationary-phase, acid-induc ed, glucose-repressed oxidative pathway and the acid -resistance pathway 2 (AR2) a stationary-phase, glutamatedependent acid-resistance (GDAR) pathway. When bacteria reach the inte stines, they are then able to traverse the colonic ep ithelium by way of M cells (San sonetti and Phalipon, 1999). At the underlying lamina propria, bacteria go on to invade macrophages. After engulfment, S. flexneri is able to lyse the phagocytic vacuole and escape into the macrophage cytoplasm. There, Shigella release the effector protei n IpaB, which triggers apopto sis via its ability to bind

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13 interleukin-1 converting enzyme (ICE), also known as Caspase-1 (Zychlinksi et al ., 1992; Chen et al ., 1996). Upon induction of apoptosis, IL-1 is released which, in turn, recruits more macrophages and dendritic cells to the site of infection (Cossart and Sansonetti, 2004). IL-1 is also a potent recruite r of polymorphonuclear leukocytes (P MNs). When PMNs arrive at the infection site, they transmigrate through the coloni c epithelial tissue to th e basolateral surface. This process causes major tissue destruction and re sults in the formation of hemorrhagic plaques or lesions as well as serving to further spread the zone of infection (S ansonetti, 2001). After escaping from macrophages, S. flexneri can then invade intestinal epithelial cells from the basolateral side by inducing cell ruffling using its type III secretion system (TTSS). Shigella s TTSS is encoded by the mxi-spa region of the virulence plasmid (Espina et al ., 2006). From inside-out, the apparatus is co mprised of a cytoplasmic bulb and a disk-like structure that spans the bacteriums inner and out er membranes. A needle like structure crosses these domains and extends outside the outer me mbrane. The needle structure mediates the delivery of several effector proteins directly from the bacterial cytoplasm into the host cell cytoplasm. The proteins that comp rise and are also transferred through Shigella s TTSS have been identified and include IpaB, IpaC, IpaD, IpaA IpgD, and VirA (Table 1-1). IpaB and IpaC form an Ipa complex at the host cell membrane surface, which has been shown to be sufficient for invasion into host cells (Mnard et al ., 1996; Table 1-1). Insertion of the IpaBC complex into the host cell membrane creates a pore and activates signal tran sduction pathways that are responsible for generating membrane ruffles. Th e IpaBC complex is also known to be necessary for enabling Shigella escape from the phagocytic vacuol e in macrophages and host cells (Page et al ., 1999). IpaB has been shown to bind to the cell surface protein CD44 and associate with 51-integrin (Lafont et al ., 2002). IpaB also eventually l eads to induction of apoptosis of

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14 Shigella -infected macrophages. Besides associating w ith IpaB to create the IpaBC complex that is necessary for entry into epith elial cells and subsequent lysis of the phagocytic vacuole, IpaC also acts to trigger F-actin nucleation vi a its C-terminal domain (Tran Van Nhieu et al ., 1999). IpaA helps to mature the entry focus induced by Shigella Once injected into the cell, it binds the actin related protein vinculin. In mammalian cells vinculin is usually found at sites of focal adhesion and is known to act as a linker between actin filaments and the plasma membrane (Tran Van Nhieu et al. 1997). Normally, vinculin exists in an au toinhibitory state in the cell due to an intramolecular association between the 95 kDa head and the 30 kDa tail domains. In this conformation, vinculins F-actin binding site contained within the tail domain is masked and, therefore, unavailable to bind actin filame nts (Johnson and Craig, 1995). While under normal conditions, an external growth hormone signal would be required to alleviate vinculins autoinhibitory state, in 1999 Bourdet-Sicard and colleagues were able to show that binding of IpaA to vinculin is able to reli eve its autoinhibitory state as well as enhance its ability to interact with F-actin. They were also able to show that the resulting complex depolymerized actin filaments both in vitro and in microinjected cells. This is in some opposition to the role usually ascribed to vinculin in mammalian cells in which it is thought that vinculin recruits VASP in cell focal adhesion sites, which can, in turn, promote actin filament assembly (Holt et al ., 1998). Nevertheless, during Shigella infection, the vinculin-IpaA comp lex can act to depolymerize actin filaments in a controlled manner at the site of bacterial entry, forming a phagocytic cup underneath the bacterium and allowing filopodial structures to extend around the bacterium, facilitating its engulfment (Bourdet-Sicard et al. 1999). IpaD is an effector protein known to be im portant in regulating secretion of other Ipa proteins as well as forming polymers with IpaC. This complex has been shown to be required for

PAGE 15

15 binding and entry of Shigella (Picking et al. 2005). Another effector protein that has been implicated in binding and entry of Shigella is IpgD (Niebuhr et al. 2000). This protein exhibits two motifs that are present in mammalia n inositol polyphosphat e 4-phosphatase and upon secretion, it specifically dephosphorylates phosph atidylinositol (4,5) bis-phosphate [PI(4,5)P2] to yield PI(5)P. PI(4,5)P2 is known to enable membrane-cytoske letal interactions via its presence in the cytosolic face of the host membrane and its ab ility to bind several actin regulatory proteins (Czech, 2000). Therefore, IpgDs action is thought to assist in relaxing membrane-cytoskeleton interactions in order to further facilitate the ex tension of actin filament s during the entry process (Niebuhr et al. 2002). Indeed, Niebuhr and colleagues we re able to show that expression of IpgD in mammalian cells led to a strong decrease in tether force pres umably by uncoupling of the plasma membrane from the unde rlying actin cytoskeleton. Shortly after being taken up by the host cell, S. flexneri is able to escape the harsh environment of the phagolysosome by inducing lysis of the vacuole and escaping into the cytosol. Epithelial cells that have been invade d release IL-8, which, in tu rn, recruits neutrophils to the site of infection. It is the presence of neutrophils that results in pus being passed in the stool, while the inflammation that ensues at th e infected site causes actual damage to the intestinal epithelium, resulting in bloody di arrhea (Niyogi, 2005). Actin-based Intraand Intercellular Motility Aside from causing damage to the intestinal epithelium, S. flexneri possesses another attribute that contributes to successful pat hogenesis. Upon escaping the vacuole and being released into the cytoplasm, S. flexneri goes on to usurp the host cells actin machinery to form rocket tails which propel the bacter ia within the cell and also allo ws direct spread to neighboring cells without being exposed to any extracellular milieu. This aspect of the pathogenesis process

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16 plays an important role in allo wing the infection to spread wi thout detection by macrophages or antibodies (Finlay and Falkow, 1997). Normally in cells, actin dynamics are meticul ously controlled by an ar ray of proteins in response to specific extracellular or intracellular stimuli (Pantaloni et al. 2001). Some key components involved in normal actin dynamics in the cell include mono meric and filamentous actin, Arp2/3, profilin, thymosin -4, N-WASP (neuronal Wisko tt-Aldrich syndrome protein), VASP (vasodilator-stimulated phosphoprotein), Rho family proteins, ADF/cofilin, and capping proteins. In non-muscle cells, the actin cyto skeleton plays key roles in whole-cell motility (Pollard and Borisy, 2003) and trafficking of intracellular organelles (Engqvist-Goldstein and Drubin, 2003). Actin exists in two forms in the cell: monomeric G-actin and filamentous F-actin. The Arp2/3 complex, which consists of the actin rela ted proteins Arp2, Arp3, a nd five other subunits, is vital in nucleating G-actin to begin F-actin formation. In or der to initiate nucleation, the Arp2/3 complex must first be activated by WASP (Wiskott-Aldrich syndrome protein) or NWASP. N-WASP exists in an autoinhibitory st ate in the cell due to sequences near the Nterminal region interacting with sequences in the C-terminal region (Bompard and Caron, 2004). In this conformation, binding domains are unava ilable, rendering N-WASP unable to interact with binding partners. It is activated and recruited to the ce ll membrane via activated members of the Rho family small GTPases such as Cdc42 a nd Rac. Once recruited to the cytosolic face of the plasma membrane, N-WASP acts as a scaffoldi ng protein that brings Arp2/3 and G-actin into spatial and functional proximity. In its unfol ded state, N-WASPs C-terminal VCA domain is made available for Arp2/3 and G-actin to bind. Nucleation by Arp2/3 leads to the formation of polarized F-actin filaments. The end where new monomers are added is termed the barbed (or

PAGE 17

17 plus) end, while the end from which older mo nomers dissociate is te rmed the pointed (or minus) end. The terms barbed and pointed refe r to previous observati ons of actin filaments decorated with myosin II S1 fragments (these fragments contain only the head and neck region of myosin II). In these experiments, the S1 fragments bind the actin filament at a 45 angle when ATP is absent, creating the appearance of a barbed end and a pointed end (Mornet et al ., 1981). The protein profilin binds G-actin monomers, ushers them onto the barbed end of the growing actin filament, and catalyzes their incorpora tion into the filament by enhancing ATP-ADP exchange on monomers (Pollard, 2007). N-WA SP and VASP contain polyproline regions to which profilin can bind (Kang et al. 1997; Suzuki and Sasakawa, 2001). It has been proposed that VASPs role is to bind the growing F-actin ch ain and bring in profilin-actin complexes to the barbed end, aiding in efficien t filament elongation (Krause et al. 2003). In order to orchestrate directional motility, capping proteins are needed to stop the growth of one or more filaments so that others may be allowed enhanced growth to propel the cell in the desi red direction. Lastly, ADF/cofilin is a protein that binds to the sides of actin filaments then severs and caps the filament, enhancing disassembly at the barbed ends. In the case of bacterial invasion, however, S. flexneri and a handful of other pathogens have each devised distinct ways of activating the hos t cells actin machinery without the involvement of external or internal signals. Specifically, S. flexneri expresses an outer-membrane protein, IcsA (or VirG), which is necessary and suffi cient (Goldberg and Theriot, 1995; Bernardini et al. 1989) for initiating its actin-based motility. IcsA is responsibl e for binding the host protein, NWASP (Loisel et al. 1999). Binding of IcsA is able to relieve the autoinhibition of N-WASP in a Cdc42-independent manner. The open conforma tion of N-WASP in turn activates the Arp2/3 complex, which is required to first stimulate nuc leation of actin filament assembly at the

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18 bacterial surface (Gouin et al. 2005). Actin filaments are assemb led at one pole of the bacterial surface with the barbed end of th e filament facing the bacterium. Subsequent addition of ATPbound monomers to the barbed end of filamentous actin generates the force necessary to propel the bacterium throughout the cytosol (Gouin et al. 2005). Using this mode of motility, S. flexneri is able to move throughout the host cell cy tosol until it reaches the inner face of the plasma membrane. Once there it is then able to generate the formation of finger-like protrusions called filopodia. These structures harbor a bacterium at their ti ps and extend from the infected cell into an uninfected neighboring cell, which su bsequently engulfs and ingests the bacteriacontaining structure (Sansonetti et al ., 1994). Once engulfed, the bacterium is able to lyse the double membrane vacuole that contains it by way of TTSS effector proteins and the cycle is repeated again (Allaoui et al ., 1992). Therefore, it is th is process that confers upon S. flexneri the ability to rapidly spread infection along the colonic epithelium. Furt her, it has been shown S. flexneri that are defective in implemen ting this process are unable to set up successful infections in animal models (Sansonetti et al ., 1991). Despite the signi ficance of this facet of S. flexneri s pathogenesis, little is understood regarding whic h factor(s) and/or proc esses are required for forming filopodia once bacteria r each the host cell periphery (Pust et al ., 2005). Filopodium Formation in Eukaryotic Cells Filopodia are defined as actin-ric h organelles that extend from the cell front in migrating cells. They play a key role in exploring new space during cell migr ation, as well as subsequently mediating adhesion to extracellular matrices or cel ls. Furthermore, several protrusion structures in specialized cells namely, lymphocyte and brush border microvilli as well as stereocilia on cochlear cells have been proposed to be rela ted to filopodia (Faix and Rottner, 2006). While it is currently unknown whether filopodia are initiate d as independent struct ures or if they are borne of preexisting cell membrane protrusions called lamellipodia, in recent years a number of

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19 molecular components have been id entified that are associated with filopodia formation. One of the main components capable of potently induci ng filopodium formation is Cdc42, a member of the Rho-family GTPases (Nobes and Hall, 1995) This phenomenon is thought to occur via direct interaction of Cdc42 with N-WASP, wh ich is a known activator of the Arp2/3 complex (Stradal et al ., 2004). However, although this complex ma y be important for nucleation of actin filaments leading to initiation of filopodium protrusion, the Arp2/3 complex is altogether absent from the tips of filopodia, indicating that furt her elongation requires the contribution of other components (Faix and Rottner, 2006). Proteins of in terest that have been shown to be present at filopodial tips include N-WASP, formins, memb ers of the Ena/VASP family, fascin, and insulin receptor substrate p53 (IRSp53). Like N-WASP, IRSp53 has been shown to be an effector protein downstream of Cdc42 (Govind et al ., 2001). Of particular inte rest, it has been proposed to contribute to efficient filopodium formation through its interaction w ith members of the Ena/Mena/VASP family (Krugmann et al ., 2001). Krugmann and colleagues (2001) were able to show that this interaction is brought about when activated Cdc42 binds to a part ial CRIB motif found within the central region of IRSp53. Binding of Cdc42 pr events an intramolecular interaction within IRSp53 and, thereby, allows the recruitment of Me na to the IRSp53 SH3 domain. Previously, it has been proposed that VASPs presence at filopodi al tips may serve to uncap the barbed ends of actin filaments, thereby encouraging mono mer addition and filopodium elongation (Bear et al. 2002; Krause et al. 2003). More recently, how ever, studies have shown that VASPs important role in filopodial formation relies heavily on its Factin bundling ability. Furthermore, these studies have also suggested that VASP does not ac t as a competitor to capping proteins nor does it depolymerize from F-actin barbed ends (Schirenbeck et al. 2005; Schirenbeck et al ., 2006).

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20 However, although VASP may play an important role in facilitating filopodium elongation, it does not have the inherent ability to drive filopodium protrusion. A family of proteins, termed diaphanous-related formins (Drf), has been shown to be involved in this facet of filopodia formation. These proteins are characterized by a highly conserved domain called the formin homology 2 (FH2) domain. This domain has been shown to possess the abil ity to nucleate and elongate unbranched actin filaments (Wallar and Alberts, 2003), creating parallel networks of actin filaments as opposed to a network in which f ilaments branch off one another at angles of 70. This characteristic is also in line with the observation that the Arp2/3 complex, which is primarily known to nucleate the branched actin filament networks mentioned above, is not found throughout the length or at the tip s of filopodia. Furthermore, Drfs are also known to be downstream effector proteins of Cdc42 (Peng et al ., 2003). In addition to protrusion, another aspect that is important in main taining filopodial integrity is th e action of stabilization of the parallel actin filaments that constitute the fil opodium. The actin-binding protein fascin serves this need by cross-linking parallel filopodial actin filaments. Fa scin is proposed to contain two actin-binding sites, one of which has been iden tified, that confer its ab ility to bundle filopodial actin filaments and thereby provide structur al stability to the filopodium (Kureishy et al ., 2002). Myosins and Filopodia Formation Myosins comprise a large superfamily of actin -dependent molecular motor proteins that perform a variety of functions in the cell. Recent research suggests they are also involved in proper formation of filopodia and similar structures. Overview of Myosins There are currently 15 distinct classes of myosins; they typi cally contain three fundamental domains that designate them as members of th e myosin superfamily: 1) a motor domain (also called a head domain) that interacts with actin and binds ATP, 2) a neck do main that is able to

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21 bind light chains or calmodulin, and 3) a tail domai n that binds specific car go(s) and/or positions the motor domain so that it can interact with actin (Sellers, 2000). Among the three domains, the motor domain is the most conserved across the 15 classes, while the neck domain and the tail domain can vary slightly and wi dely, respectively. Myosins can exist as single molecules or as homodimers. The possibility of dimerization is usually depend ent upon the presence of a coiled coil region in the neck domain. There is no predetermined criteria stating which region(s) constitutes a myosin tail domain. Across the 15 classes, this domain is the most divergent and is usually the domain that confers upon each class of myosin its unique functionality. Class II myosins are predominantly functional in muscle cells and were the first class of myosins to be identified. For several decades they were the only class thought to exist and it is for this reason that they are so metimes referred to as conven tional myosins (Sellers, 2000). Hence, all myosin classes that were discovered su bsequently are referred to as unconventional myosins. The functions of unc onventional myosins are manifest ed in many cellular processes including membrane trafficking, cell move ments, and signal transduction (Mermall et al ., 1998). It has even been shown that cert ain myosins play a key role in several sensory systems, including hearing, balance, and vision. Of note is the fi nding that myosins Ic, VI, VIIa, XVa, and others are necessary for the proper formation of ster eocilia (microvilli-like cell projections found on hair cells in the inner ear). Mutations in these genes have been shown to be the cause of hearing loss and some balance diso rders in vertebrates and Drosophila melanogaster (Frolenkov et al ., 2004). This is one specific example that illust rates the involvement of myosins in the proper formation and/or extension of actin-rich protrusions. Myosin-X (M10) and Filopodia Formation Myosin-X (M10) is a recently discovered unconventional myosin that is found ubiquitously, though in low quantities, in various tissue types (Berg et al ., 2000). Although its

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22 structure has not yet been determined, M10 is proposed to be a double-headed myosin motor due to the presence of a putative coiled coil domain. Each tail domain contains three total pleckstrin homology (PH) domains (only tw o of which are thought to be functional), a myosin tail homology 4 (MyTH4) domain, and a band 4.1/ezrin/radixi n/moesin (FERM domain). This unique tail composition makes it the founding a nd sole member of its class (Sellers et al ., 2000). In 2002, Berg and colleagues were able to show that overexpression of full-length M10, but not M10 truncates, was able to increase the number a nd length of filopodia formed in HeLa cells. Furthermore, by using a GFP-M10 construct, they were also able to demonstrate that M10 specifically localizes to the tips of filopodia and undergoes fo rward and rearward movement within the filopodia. This observation led them to suggest that M10 acts as an intrafilopodial motor that may have a role in de livering cargo(s) to filopodial tip s. While the issue of whether or not M10 does in fact play a ro le in transporting cargo in the cell as well as the identity of possible cargos remains to be resolved, ther e have been numerous reports of M10 being implicated in various cellular processes. Besides filopodia formation, other cellular proce sses in which M10 has been shown to play a role include nuclear anchori ng, spindle assembly, and axonal growth cone guidance (Weber et al ., 2004; Zhu et al ., 2007; Toyoshima and Nishida, 2007). In 2004 it was demonstrated that the MyTH4-FERM domain of M10 was able to bind direc tly to purified micr otubules and disruption of M10 function led to a disruption in nuclear anchoring, spindle assembly, and spindleF-actin association during meiosis (Weber et al ., 2004). In a similar vein, in 2007, Toyoshima and Nishida were further able to show that M10 was involved in properly orienting the mitotic spindle parallel to the substrat um in nonpolarized culture cells in a manner that was dependent on integrin mediated adhesion. They were able to show that disruption of adhesion via pathways

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23 that interfere with integrin-mediated adhesion caused misorientation of the mitotic spindle. Furthermore, when they knocked out M10 using si RNA, they found that spindle orientation was impaired much the same as it was when integr in-mediated adhesion was obstructed. In addition to their findings that nonpolarized culture cells expressing normal M10 levels plated on culture dishes coated with poly-L-lysine (a coating th at precludes integrin-m ediated adhesion) also exhibited aberrations in mitotic spindle orientation, they were also able to successfully demonstrate at least one other instance wher e M10 function was inhibited when upstream integrin-mediated signaling was interrupted. In their work, Toyoshima and Nishida referred to Weber et al .s findings in 2004 that cited M10s ability to directly bind microtubules as a means by which it could affect mitotic sp indle orientation. Aside from its ability to interact with microtubules, it has also been proposed that M1 0s PH2 domain may serve to properly target M10 to the membrane in response to agonists th at activate phosphatidyli nositol 3-kinaase (PI3K) by virtue the domains ability to bind phos phatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] (Tacon et al ., 2004). However, this statement is in some opposition to Berg and Cheneys observations that GFP-M10 constructs that were missing the head (motor) domain did not exhibit proper localization to f ilopodial tips, but exhibited a diffuse localization pattern instead (Berg and Cheney, 2002). In addition to induction of filopodia, nuclear anchoring, and proper spindle formation, M10 has also been shown to play a role in nervous system development. Many other myosins have also been previously demonstrated to play a role in nervous or sensory system development owing to their roles as for ce-producing modulators of the ac tin cytoskeleton (Brown and Bridgman, 2004). Specifically, in 2007, Z hu and colleagues pub lished a report that demonstrated M10s involvement in axonal pa th-finding. They found M10 plays an important

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24 role in properly distributing netr in receptors in neuronal cells in order to correctly respond to netrin-1 cues during the process of neur ite outgrowth. This was demonstrated in vitro when silencing of M10 prevented pr oper distribution of netrin receptors in neurites and in vivo by showing that expression of a motor-less (nonfunctional) M10 was able to reduce neurite outgrowth in response to netrin-1 in cortical explants derived fr om mouse embryos. This is one of several instances in which it has been shown that MyTH4-FERM myosins play an important role in maintenance of cellular proj ections in specialized cells (Oliver et al. 1999).

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25 Table 1-1. List of S. flexneri TTSS e ffector proteins and their functions Effector protein Function IpaA Binds host cell vinculin to help depolymerize actin and mature phagocytic cup during bacterial entry IpaB Binds cell surface protei n CD44 and associates with 51-integrin Leads to induction of apoptosis in macrophages Binds to IpaC to form Ipa complex that is embedded into host cell membrane IpaC Binds to IpaB to form Ipa complex that is embedded into host cell Membrane Triggers F-actin nucleation via C-terminal domain IpaD Known to form polymers with IpaC Known to be needed for binding and entry of Shigella IpgD Specifically dephosphorylates PI(4,5)P2 to yield PI(5)P Thought to aid in relaxing membra ne-cytoskeleton interaction to facilitate actin fila ment extensions during entry VirA Triggers host microtubule dest abilization and leads to membrane ruffling during bacterial entry (Yoshida et al ., 2002)

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26 Table 1-2. List of cytoskeletal co mponents found in mammalian cells Cytoskeletal component Role Actin 47 kDa globular protein that can exist as monomers (G-actin) or can be polarized into filaments (F-actin) in the cell. Structural protein for the microf ilament layer of mammalian cell cytoskeleton Arp2/3 complex Complex comprised of actin -related protein ARP 2, ARP3, and five other subunits that is necessary for de novo nucleation of new actin filaments in the cell. Binds to the side of an existing actin filament and initiates growth of a new filament at a 70 angle to the existing filament; leads to the establishment of branch ed actin networks in the cell. Profilin Binds G-actin monomers and ushers them to the barbed end of a growing actin filament. Enhances ATP-ADP exchange when new monomers are incorporated into a growing actin filament. N-WASP Activates the Arp2/3 complex and acts as a scaffolding protein to bring Arp2/3 and G-actin into spatial and functiona l proximity to initiate the formation of actin filaments. VASP Simultaneously binds the growi ng F-actin filament and profilinactin complexes to aid in efficient filament elongation. Rho family proteins GTPases that, once activated in response to extracellular signals, activate and recruit N-WASP to the cytosolic face of the plasma membrane. ADF/cofilin Bind to the sides of actin filaments and sever filaments to enhance disassembly at the barbed end. Capping proteins Bind to the barbed end of actin filaments to stop the growth of one or more filaments so that others may be allowed enhanced growth to propel the cell in the desired direction. Thymosin -4 Sequesters actin monomers in orde r to maintain a cytoplasmic pool of free actin monomers, which can then be used for rapid filament elongation of F-actin (Dedova et al. 2006).

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27 CHAPTER 2 METHODS AND MATERIALS Cell Infections with S. flexneri and M10 Localization Cell Cultures and Bacterial Strains HeLa (human cervical cancer) cells, PtK2 (kangaroo rat kidney) cells, and Cos7 (African green monkey kidney) cells were maintained at 37C and 5% CO2 in Dulbeccos Modified Essential Media (DMEM) containing 10% fetal bovine serum and 5% penicillin/streptomycin antibiotic solution (DMEM complete). S. flexneri strain 2457T, a virulent strain of serotype 2a (Wei et al ., 2003), was a kind gift from Dr. Marcia Goldberg, Massac husetts General Hospital. Individual bacterial colo nies were selected from a tryptic so y agar or brain heart infusion agar plate containing 0.01% Congo Re d dye to ensure bacterial virulence (Meitert et al ., 1991). Colonies were inoculated into tryptic soy brot h (TSB) or brain heart infusion (BHI) and were grown overnight in a shaker at 37C. The ne xt day, a diluted culture was made from the overnight culture and allowed to grow to an OD600 between 0.600 0.800 to ensure optimal expression of the outer-membran e protein, IcsA (Gold berg et al ., 1994; Stevens et al ., 2006). For experiments requiring Listeria monocytogenes bacteria were inoculated into BHI and grown overnight in a shaker at 37C. Transfection with cDNA Plasmids Transfections with various M10 cDNA plas mid constructs, an Akt-PH cDNA plasmid construct, and a PLC -PH cDNA plasmid (Figure 3-4) were accomplished using FuGene6 Transfection Reagent (Roche) or Lipofec tin Reagent (Invitrogen) according to the manufacturers instruct ions. M10 plasmid constructs that were used were constructed as described previously (Berg and Cheney, 2002; Bohil et al ., 2006): full-length green fluorescent protein-tagged (GFP)-M10, GFP-M10-HMM (incl udes head, neck, and proposed coiled-coil

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28 domains), GFP-M10FERM (full-length M10 with the FE RM domain deleted), and GFP-M10MyTH4 FERM (full-length M10 with the MyTH4 and FERM domains deleted). The GFPAkt-PH plasmid (contains the PH domain of th e Akt protein kinase, amino acids 1-167) and GFP-PLC -PH plasmid (contains the PH domain of PLC amino acids 1-170) was constructed as described previously (Vrnai and Balla, 1998). Cells were allo wed to incubate at 37C and 5% CO2 overnight to allow for uptake and expression of cDNA. Cell Invasion S. flexneri invasion of semiconfluent cell monol ayers was performed as described previously with some modifications (Zeile et al ., 1996). Briefly, bacteria in log phase were spun down, washed, and resuspended in 1X PBS. Bacter ia were then added to HeLa cells or Cos7 cells grown on 35-mm culture dishes at an MO I of 50 bacteria per cell and subsequently centrifuged at 500x g for 15-30 minutes to allow for bacterial adhesion and entry. Culture dishes were then incubated for another 30-90 minutes at 37C and 5% CO2 to allow for initiation of bacterial actin-based motility and filopodia formation. Extracellula r bacteria were then removed by washing cells with 1X PBS. Media containing gentamicin at a concentration of 10g/mL was then added back to the culture dishes to preven t growth of extracellular bacteria and cells were incubated at 37C and 5% CO2 for 10-15 min before being viewed via video microscopy. L. monocytogenes invasion of semiconfluent HeLa cell monolayers was carried out as described previously (Sidhu et al ., 2005). Immunofluorescence and Phalloidin Stain For immunofluorescence experiments, HeLa cells infected with S. flexneri were treated with 3.7% formaldehyde, then permeabilized with 0.2% Triton X-100. A blocking solution (5% fetal bovine serum resuspended in 1X PBS) wa s then added to cells, followed by primary antibody at a concentration of 1:250 (tropomyos in monoclonal mouse antibody was purchased

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29 from Sigma and M10 polyclona l rabbit antibody was a kind gift from Dr. Richard Cheney, University of North Carolina at Chapel Hill). Lastly, a FITC-conjugated secondary antibody was added. For experiments where F-actin filame nts were stained with phalloidin (Molecular Probes), phalloidin resuspended in blocking solu tion at a concentration of 1:400 was added to cells following the permeabilization step. LY294002 Treatment For experiments using a PI3K inhibitor, LY 294002 resuspended in serum free media (final concentration 50M) was added to Shigella -infected cells one hour afte r initiation of infection and left to further incubate at 37C and 5% CO2 for another 45-60 min to allow sufficient time for formation of filopodia. At this point, cells were washed and media containing LY294002 and gentamicin (10g/mL) was added back to cells. Cells were allowed to incubate at 37C and 5% CO2 for an additional 10-15 min before filopodia were analyzed via video microscopy. Video Microscopy Time-lapse (live) and immunofluorescence (fix ed) images were obtained using either a Nikon (Tokyo, Japan) or Zeiss (Germany) inverted microscope connected to a cooled chargecoupled device camera (Hamamatsu, model C5985). Images were analyzed using Metamorph 4.0 image software (Universal Imaging, West Ch ester, PA) or AxioVision Release 4.6 image software (Carl Zeiss MicroImaging, Inc.). Transfection with siRNAs Transfections with control siRNA (purchased from either Qiagen or Dharmacon) and M10 siRNA (purchased from Qiagen as described previously by Zhang et al ., 2004 or purchased from Dharmacon as catalog number J-007217-06-0005) were accomp lished using the RNAifect Transfection Reagent (Qiagen) acco rding to the manufact urers instruct ions. Cells were allowed

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30 to incubate at 37C and 5% CO2 for 48 h to allow for efficient knockdown of M10. A Western blot was performed as described below to confirm knockdown of endogenous M10. Western Blots Western blots were performed using cytopl asmic extracts subjected to SDS-PAGE. Confluent monolayers of either HeLa, Cos7, or Caco2 cells were washed once and subsequently scraped in a 150l volume of 1X PBS containi ng 10x protease inhibitor cocktail (Complete Protease Inhibitor Cocktail tablets, Roche). The solution was then passaged several times through a 23or 25-guage needle to lyse cells and shear DNA without causing proteolysis to endogenous M10 levels. Samples we re loaded onto a 7.5% polyacry lamide gel, then proteins were transferred to a PVDF membrane (Millipor e). A polyclonal M10 antibody raised in rabbit was used at a dilution of 1:1000 to probe th e membrane (M10 antibody was provided by Dr. Richard Cheney, University of North Carolina at Chapel Hill) followed by an HRP-conjugated secondary antibody at a dilution of 1:2000. Super Signal West Pico Chemiluminescent Substrate was used for protein detection (Pierce). To verify equal protein lo ading, the membrane was stripped using Restore Western Blot Stripping Buffer (Pierce) and re-blotted with monoclonal actin antibody raised in mouse (Sigma) at a dilution of 1:5000 followed by an HRP-conjugated secondary antibody at a dilution of 1:10000. Plaque Assays The plaque assay protocol was carried out as previously described (Oaks et al ., 1985), with some modifications. Briefly, HeLa cells were grown to confluency in 6-well plates containing appropriate media at 37C and 5% CO2. Cells were then treated with either control siRNA or M10 siRNA (Qiagen and Dharmacon) and allowe d to incubate for 48 h at 37C and 5% CO2 to allow for efficient knockdown of M10. On the day of infection, S. flexneri grown to log phase were centrifuged, washed, and resuspended in 1X PBS. Bacteria were added to cell monolayers

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31 at an MOI of 5 bacteria per cell. Plat es were then incuba ted at 37C and 5% CO2 for 90 minutes. During this adsorption or attachment period, plates were rocked back and forth every 30 minutes to ensure equal distribution of b acteria over cell monolayers. Next an agarose overlay (2 mL per well) consisting of appropriate media (serum-c ontaining DMEM for HeLa cells or appropriate serum-containing MEM for Caco2 cells), 10 g/mL of gentamicin, and 5% low-melting temperature agarose (Fisher Scientific) was adde d to each well. Plates were left at room temperature in a tissue culture hood for 10-15 minutes to allow for the agar ose overlay to solidify and then incubated overnight at 37C and 5% CO2. The next day, a secondary agarose layer (1 mL per well) consisting of appropriate media, 10 g/mL of gentamicin, 5% low-melting temperature agarose, and 0.1% neut ral red dye (Sigma) to assist in visualization of plaques was added to each well. Plates were then incubated at 37C and 5% CO2 for 24-48 hours and then examined for the formation of plaques.

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32 CHAPTER 3 RESULTS M10 Localizes to S. flexneri Contained in Filopodia Introduction In eukaryotic cells, filopodium forma tion requires both actin polymerization and reshaping of the plasma membrane. Since Shigella s intracellular motility is dependent upon actin polymerization, it seems plausible that one or more actin-plasma membrane linker proteins may be involved in the process of filopodium form ation that occurs subse quently. In relation to our research interests, M10 stands out as a potential can didate in this facet of Shigella s pathogenicity because it has previously been show n to be an important factor in filopodium formation in eukaryotic cells (Berg et al ., 2002). It has also been shown that although actin polymerization alone is necessary to drive vari ous forms of eukaryotic cell motility, myosins can oftentimes be implicated in powering certain processes along with actin (Berg et al ., 2001). Some such processes include signal transduction (Bahler, 2000) and establishment of polarity (Yin et al., 2000). It is known that filopodium formation me diated by IcsA and subsequent uptake by adjacent epithelial cells is an important facet for virulent Shigella to establish a successful infection in animals (Sansonetti et al., 1991). Since to date little is known about the mechanism(s) Shigella undertakes to carry ou t this process, we sought to investigate whether M10 played a role and, if so, wh ether its role could shed any li ght on the process as a whole. Firstly, it was necessary to determine the presence or ab sence of M10 in Shigella -laden filopodia. We employed a GFP-M10 plasmid construct in orde r to visualize the loca tion of wild type M10 in live cells during infection with S. flexneri and utilized immunofluor escence techniques to determine the location of endogenous M10 in fi xed cells that had been infected with S. flexneri

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33 M10 localizes to motile Shigella but not motile Listeria in HeLa cells A number of bacterial species such as S. flexneri L. monocytogenes and several Rickettsia species are able to invade non-phagocytic host cells and subsequently usurp host cell actin machinery and undergo actin-based motility (Gouin et al ., 2005). While the end effect of actin polymerization, namely a means of propulsi on within the host cell, is the same for the various bacterial species that u tilize it, the mechanisms by which actin polymerization is initiated varies from pathogen to pathogen. Specifically, S. flexneri activates the Arp2/3 complex to initiate actin assembly indirectly by fi rst binding and activating N-WASP, whereas L. monocytogenes is able to directly bind and activat e the Arp2/3 complex via ActA (Kocks et al ., 1992; Gouin et al ., 2005). This difference in mechanisms for achieving the same goal of initiating actin-based motility for both pathogens s uggests that other differences may also exist in latter parts of the motility process. By obser ving HeLa cells expressing a full-length GFP-M10 plasmid construct using video microscopy, we found that during infec tion M10 was localized most notably along the side s of filopodia containing Shigella (figure 3-1A) and sometimes in actin tails of motile Shigella Under the same experimental co nditions we found that M10 failed to localize to any Listeriainduced actin structures (figure 3-1B). To further verify these findings, we performed immuno fluorescence experiments on HeLa cells infected with S. flexneri These experiments confirmed that endogenous M10 wa s concentrated behind, as well as partially alongside, bacteria that were found in filopodia and that some intracellular bacteria recruited M10 at one pole (figure 3-2). Th ese observations indicate that at least one host factor may be involved in some aspects of mo tility and filopodium formation in S. flexneri pathogenesis, but not in L. moncytogenes pathogenesis.

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34 Tropomyosin localizes to Listeria -Induced, but not Shigella -Induced, Actin Tails Previously, it had been noted that the ac tin-binding protein, tropo myosin, localized to Listeria -induced actin structures (Dabiri et al ., 1990). However, there are no documented reports of whether or not tropomyosin is recruited to Shigellainduced actin tails. Tropomyosin is able to bind along the sides of polymerized actin filaments and, once bound, preclude the binding of myosin heads to the actin f ilament (Cooper, 2002). We hypothe sized that one reason why we were observing M10 localization in Shigella -induced, but not Listeria -induced, actin tails was because of the possibility that tropomyosin was absent from actin tails induced by Shigella Indeed, when Shigella -infected HeLa cells were co-stained with phalloidin and a tropomyosin antibody, there was no tropomyosin lo calization to bacteria-induced actin tails. This was in contrast to Listeria -infected HeLa cells in which co-stain ing with phalloidin and a tropomyosin antibody almost always revealed co-localizatio n (figure 3-3). While additional factors and processes may also be at play, we concl uded that the presence of tropomyosin in Listeria induced actin tails, and its absence from Shigella -induced actin tails, provides one clue as to why M10 is recruited to the actin tails of the la tter while being excluded from the former. Tail Region of M10 Facilitates Shigella s Ability to Form Longer Filopodia Introduction Myosin-X is a 235 kDa protein that is charac terized by a head (motor) domain that shares <45% identity with other myosin s, 3 IQ motifs, and a predicted coiled-coil region (suggests that native M10 exists as a dimer in cells). The ta il domain consists of a MyTH4 domain, a FERM domain, and 3 PH domains (Berg et al. 2000). In 2002, Berg and Cheney discovered that M10 localizes to the tips of filopodia and undergoes forw ard and rearward motion within filopodia. In order to target which region(s) conferred the proteins ability to localize to the filopodial tips, they generated several GFP plasmid constructs : GFP-M10, the full-length protein with a GFP

PAGE 35

35 tag; GFP-M10-HMM, a heavy meromyosin (H MM)-like fragment which contains the head, neck, and coiled coil regions; a nd GFP-M10-Tail, which constitu tes the distal tail domain including the PH domains, MyTH4 domain, and the FERM domain. Their findings revealed that both the GFP-M10 and the GFP-M10-HMM constructs were able to localize to the tips of filopodia in a pattern similar to that seen when using immunofluorescence to visualize endogenous M10. They concluded, th erefore, that the motor, or head, domain of M10 was the region responsible for properly targeting M10 to filopodial tips and speculated that the various domains within the tail region perh aps contribute to the transport of cargo of some kind. At this point it remains unclear which cargo(s) may speci fically be transported by the domains found in M10s tail region, but several invest igations have identified certai n cellular binding partners for these domains. For example, the second of the three PH domains has been shown to bind the PI3K product, PI(3,4,5)P3 (Isakoff et al ., 1998; Tacon et al ., 2004). PI(3,4,5)P3 is known to function as a second messenger molecule that ac ts to induce local actin polymerization (Insall and Weiner, 2001). Furthermore, M10 was recently shown to be a downstream effector of PI3K during phagocytosis in macrophages, demonstrating at least one functional role for M10 in cells (Cox et al ., 2002). Immediately following the last PH domain is a myosin tail homology 4 (MyTH4) domain. This domain is also found in class VII myosins, several unconventional myosins (M4, M10, M12, and M15), and kinesinlike calmodulin binding protein (KLCBP), which is a microtubule binding moto r protein found in plants (Reddy et al ., 1996; Oliver et al ., 1999). At this point in time, the MyTH4 domain has been shown to have the ability to bind microtubules only in KLCBP (Narasimhulu and Reddy, 1998). Finally, the most C-terminal domain found in M10 is the FERM domain. The domain is named for a group of cytoskeletonmembrane linker proteins (ba nd 4.1/ezrin/radixin/moesin) in which the domain was first

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36 identified and attributed the function of having the ability to bind PI(4,5)P2, a molecule present in the inner leaflet of th e plasma membrane (Chishti et al ., 1998; Tacon et al ., 2004). Despite this function among the traditional FERM pr oteins, the FERM domain found in M10 does not bind PI(4,5)P2, but has been shown to bind -integrins (Zhang et al ., 2004). The ability of M10 to bind -integrins has been shown to be important in cell processes such as integrin-dependent adhesion and filopodial extension as well as neurite outgrowth a nd growth-cone guidance (Zhang et al ., 2004; Zhu et al ., 2007). Absence of M10 Tail Region Results in Shorter Shigella -Induced Filopodia To determine which of the M10 ta il region domains was important for Shigella filopodia length, we transfected Cos7 cells with one of several GFP-M10 constructs -GFP-M10FERM, GFP-M10MyTH4 FERM, and GFP-M10-HMM and compared Shigella -induced filopodia lengths in these cells to contro l cells transfected with full-len gth GFP-M10. We opted to use Cos7 cells because they contain a low bac kground level of endogenous M10 (figure 3-5) and they naturally do not express filopodia on their surface (Bohil et al ., 2006). These conditions would enable us to assess the effects of the exogenously expressed M10 constructs with minimal interference from the native M10 population. Lengths of Shigella -induced filopodia in cells transfected with GFP-M10-HMM were compared to cells transfected with GFP-M10. A 33% decrease in average filopodial lengths was observed in GFP-M10HMM transfected cells (figure 3-6). This observation led us to conjecture th at the tail region of M10 was important for Shigella s ability to form filopodia. This is not surprising, since the tail is the region where various domains are found to which specific bind ing partners can bind and confer, in part, diverse functionality am ong myosins (Krendel and Mooseker, 2005).

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37 The MyTH4 and FERM Domains of M10 Are Not Required for Efficient Shigella -Induced Filopodium Formation Although little is known about which cellular compone nts are required in the process of filopodial formation for Shigella there have been some observations noted for both Shigella and Listeria another intracellular pathogen that usurps host cell actin and forms filopodia into adjacent cells to disseminate during infection (Carlsson and Brown, 2006). In 2005, Pust and colleagues reported that the host cel l protein ezrin, a member of the family of proteins containing a FERM domain, accumulated at the sites of Listeria -induced protrusions a nd were further able to show that disruption of its ability to bi nd CD44, an integral membrane protein, hampered Listeria s ability to efficiently form filopodia. For Shigella it has previously been shown that the inhibition of myosin light chain kinase (M LCK) and, presumably, the inability of myosin II to be phosphorylated leads to a marked decrease in cell to cell spread in Caco 2 cells (Rathman et al ., 2000). In both instances, it is the disruption of an involved host cell factor that is responsible for the impairment of intercellular bacteria l spread. Furthermore, in the case of Shigella the contribution of a myosin is implied in the proces s. With this in mind, as well as the knowledge that M10 contains a FERM domain, we proceeded to transfect Cos7 cells with either GFP-M10FERM or GFP-M10MyTH4FERM and compare the resulting Shigella -induced filopodial lengths to a control (Cos7 cells transfected with GFP-M10) to see if we could reproduce the results from our experiment w ith GFP-M10-HMM and thereby identify the domain responsible for facilitating proper filopodial length. We f ound that transfections with either GFP-M10FERM or GFP-M10MyTH4 FERM did not yield Shigella -induced filopodia that were shorter in length than those formed with full-leng th GFP-M10 transfected cel ls (figures 3-7A and 3-7B). These results led us to hypothesize th at the required domain for proper filopodium formation was the PH domain.

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38 Inhibition of PI3K products and overex pression of GFP-Akt-PH Do Not Affect Shigella Induced Filopodial Lengths PH domains are found in over 100 different ce llular proteins and are characterized by sequence similarity to two regions in pleckstrin, a major substrate of protein kinase C in platelets (Kavran et al ., 1998). Proteins containing PH domains ar e involved in various tasks in the cell, including signaling processes, cytoskeleton orga nization, regulation of intracellular membrane transport, and modification of membrane phospholipids. The t ypes of proteins in which PH domains are found are varied as well and can usually be sorted into groups based on functionality. These groups of pr oteins include: Serine/Threoni ne protein kinases, Tyr protein kinases, small G-protein regulat ors, endocytic GTPases, adapto rs, phosphoinositide metabolizing enzymes, and cytoskeletal associated proteins (Rebecchi and Scarlata, 1998). Two relatively well-studied PH domain s are that of PLC1, a member of the phospholipase C family that hydrolyzes PI(4,5)P2, and that of Akt, a proto-oncogenic se r/thr kinase. While the PH domain of PLC1 specifically binds PI(4,5)P2 (Pawelczyk and Lowenstein, 1993), the PH domain of Akt preferably binds phosphatidyli nositol products of PI-3 kinase (PI3K) such as PI(3,4,5)P3 and PI(3,4)P2 (James et al ., 1996; Franke et al ., 1997). Binding of PH domains in these and other proteins to various phosphatidylinositol phospholip ids in the plasma memb rane and intracellular membranes is usually for the purpose of regulating protein activity and/or ta rgeting the protein to its required intracellular location (Lemmon and Ferguson, 2001; Tacon et al ., 2004). Since neither the GFP-M10FERM nor the GFP-M10MyTH4 FERM constructs had any effect on Shigella -induced filopodial lengths even t hough lengths were shorter when the GFP-M10-HMM construct was used, we hypothesized that one or more of the PH domains in the M10 tail region was responsible for th e role M10 was playing in enabling Shigella to efficiently form filopodia. It has previously been s hown that the M10 PH2 domain binds PI(3,4,5)P3

PAGE 39

39 (Tacon et al ., 2004). With this in mind, we sought to abrogate this domains functionality using two approaches first, we c hose to specifically inhibit PI 3K, by whose activity PI(3,4,5)P3 is a product, by using the reversib le inhibitor LY294002 (Vlahos et al ., 1994) and second, we overexpressed the PH domain of Akt, which is known to bind PI(3,4,5)P3, as a way to compete out the native binding partner for the M10 PH2 domain. We wanted to use two approaches since we werent sure whether the bind ing partner was a host cell factor or a bacterial component. We speculated that if the binding partner was a host ce ll factor, then both approaches would result in filopodia of shorter length similar to the results we obtained when we transfected cells with GFPM10-HMM. However, if it was a bacterial component, then inhi biting PI3K activity might not have any effect on filopodial length while overexpression of GFP-Akt-PH should compete out the native binding partner no matter where it originat ed. Surprisingly, when HeLa cells infected with Shigella were either transfected with GFP-Ak t-PH or treated with LY294002, there was no significant change in Shigella -induced filopodial lengths observed (figures 3-8A 3-8C). To rule out the involvement of other phosphatidyl phosphoinositide groups, we also repeated these experiments in HeLa cells transfected with GFP-PLC -PH. In these experiments, filopodia formed from cells transfected with GFP-PLC -PH did not differ in length from filopodia formed from cells transfected with GFP only (figure 3-8C ). These findings seem to indicate that there may either be redundant factors that can compen sate for the abrogation of M10s PH2 domains functionality or that the presence of one of th e other PH domains within the M10 tail region is able to mediate the functionality required for Shigella to form filopodia.

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40 Reduction of Endogenous M10 Levels Curtails S. flexneri Cell-to-Cell Spread in HeLa Cell Monolayers In Vitro In order to establish a successful infe ction in the host, it is necessary for Shigella to be able to move from an infected cell to neighboring cells along the colonic ep ithelium (Sansonetti, 2001). This process of in tercellular advancement by Shigella causes the formation of hemorrhagic plaques within the colonic epithelium in the infected host. There are a few experimental procedures that have been devised to test Shigella s virulence capacity in this aspect. Two such tests include the Sereny test (k eratoconjunctivitis shigellosa), in which virulent bacteria are placed on the conjunctiva of the gui nea pig eye resulting in a rapid spread of keratoconjunctivitis (Sereny, 1955) and a cult ured cell monolayer plaque assay (Oaks et al ., 1985), in which virulent bacteria are allowed to infect a conflu ent monolayer of cultured HeLa cells. The plaque assay requires an agarose ove rlay on the cultured cell monolayer so that the formation of plaques can be observed after a few days post-infection. It is known that virulent S. flexneri strains invade HeLa cells with high effici ency and those strains that are capable of undergoing cell-to-cell spread in cultured cell monolayers ofte n lyse or otherwise kill the infected cells leaving behind a zone of clearing (i.e., plaq ues) in the monolayer (Oaks et al ., 1985; Sansonetti et al ., 1986). In order to further assess M10s importance in Shigella s ability to efficiently form filopodia and thereby carry out effi cient intercellular sp read, we decided to test what effect knocking down endogenous M10 levels using siRNA would have on Shigella s ability to form plaques on a cultured HeLa cell monolayer. When compared to HeLa cell monolayers treated with a non-targeting control siRNA, Shigella were less able to form plaques in HeLa cell monolayers treated with M10 siRNA to knock do wn endogenous M10 levels. We found there were 26% fewer plaques formed in M10 siR NA-treated HeLa cell monolayers compared to

PAGE 41

41 control siRNA-treated monolayers (figure 3-9). These observati ons are consistent with our previous observation that Shigella -induced filopodial lengths meas ured in HeLa cells transfected with M10 siRNA were 30% shorter, on average, compared to those measured in HeLa cells transfected with ctrl si RNA (Figure 3-10). Figure 3-1A. Time-lapse pictures of GFP-M10 localizing to motile intracellular Shigella in PtK2 cells. Pictures were taken at 20s intervals. Arrows indicate nodules of moving GFP-M10 alongside motile Shigella.

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42 Figure 3-1B. Time lapse pictures of PtK2 cells transfected with GFP-M10 and infected with Listeria. GFP-M10 does not localize to motile intracellular Listeria. Arrows indicate motile intracellular Listeria. A rrowheads indicate normal localization of GFP-M10 in host cell filopodial tips.

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43 Figure 3-2. M10 antibody localizes to intracellular Shigella and Shigella-laden filopodia. A, C, and E are phase images. B, D, and F are corresponding immunofluorescence images.

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44 Figure 3-3. Tropomyosin monoclonal antibody locali zes to Listeria-, but not Shigella-induced actin tails in HeLa cells. A and B depict Listeria-infected ce lls; C and D depict Shigella-infected cells (green FITC phalloidin; red tropo myosin monoclonal antibody).

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45 Figure 3-4. The GFP-tagged cDNA cons tructs used in cell transfections.

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46 Figure 3-5. Western blot showing endogenous M10 le vels in Cos7 cell extracts. Lane labeled Cos7 extract represents protein conten t of non-transfected Cos7 cells. Lane labeled Cos7 extract + GFP-M10 repres ents protein content of Cos7 cells transfected with GFP-M10. 75kDa band represents an unknown protein with which the M10 antibody cross-reacts a nd serves as a loading control. Figure 3-6. Cos7 cells were transfected with either full-length GFP-M10 or GFP-M10-HMM, infected with Shigella, and analyzed using video microscopy. The average filopodial lengths are repr esented. On average, Shigella formed filopodia that were 33% shorter in Cos7 cells transfected with GFP-M10-HMM compared to Cos7 cells transfected with GFP-M10 (11.66 m compared to 15.25 m, respectively. P value is 0.006). For GFP-M10 (black bar) and GFP-M10-HMM (gray bar), n = 24 and 44, respectively. Data is representative of three independent experiments.

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47 Figure 3-7A. Cos7 cells were transfected w ith either full-length GFP-M10 or GFP-M10FERM, infected with Shigella, and analyzed using video microscopy. The average filopodial lengths are represente d. There was no significant difference when Shigella-induced filopodial lengths from Cos7 cells transfected with GFPM10FERM were compared with those from Cos7 cells transfected with GFPM10 (11.10 m compared to 12.22 m, respectively. P value is 0.54). For GFPM10 (black bar) and GFP-M10FERM (gray bar), n = 21 and 27, respectively. Data is representative of th ree independent experiments.

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48 Figure 3-7B. Cos7 cells were transfected w ith either full-length GFP-M10 or GFP-M10MyTH4 FERM, infected with Shigella, and analyzed using video microscopy. The average filopodial lengths are re presented. There was no significant difference when Shigella -induced filopodial lengths fr om Cos7 cells transfected with GFP-M10myTH4 FERM were compared with those from Cos7 cells transfected with GFP-M10 (12.76 m compared to 12.77 m, respectively. P value is 0.65). For GFP-M10 (black bar) and GFP-M10FERM (gray bar), n = 36 and 26, respectively. Data is representative of three independent experiments.

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49 Figure 3-8A. HeLa cells were infected with Shige lla, treated with either DMSO (control) or LY 294002, and analyzed using video microscopy. The average filopodial lengths are represented. There was no si gnificant difference when Shigella -induced filopodial lengths from DMSO-treated HeLa cells were compared with LY294002-treated HeLa cells (13.88 m compared with 15.00 m, respectively. P value is 0.49). For DMSO-treated cells (black bar) and LY 294002-treated cells (gray bar), n = 45 and 54, respectively. Data is representative of three independent experiments.

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50 Figure 3-8B. HeLa cells were either transfected with GFP-Akt-PH or not transfected (control), infected with Shigella, and analyzed using video microscopy. The average filopodial lengths are repr esented. There was no si gnificant difference when Shigella -induced filopodial lengths from HeLa cells transfected with GFP-Akt-PH were compared with those from non-transfected HeLa cells (12.30 m compared with 12.27 m, respectively. P value is 0.97). Fo r cells transfected with GFP-AktPH (black bar) and non-transfected cells (gray bar), n = 26 and 50, respectively. Data is representative of th ree independent experiments.

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51 Figure 3-8C. HeLa cells were ei ther transfected with GFP-PLC -PH or GFP only, infected with Shigella and analyzed using video microsc opy. The average filopodial lengths are represented. There was no significant difference when Shigella -induced filopodial lengths from HeLa ce lls transfected with GFP-PLC -PH were compared with those transfected with GFP only (16.12 m compared with 15.54 m, respectively. P value is 0.59). Fo r cells transfected with GFP (black bar) and GFP-PLC -PH (gray bar), n = 43 and 36, respectively. Data is representative of three independent experiments.

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52 Figure 3-9. Bar graph showing number of plaques formed on HeLa cell monolayers by S. flexneri HeLa cells were treated with e ither control siRNA or M10 siRNA. Western blotting revealed ~80% reduc tion of endogenous M10 in HeLa cells treated with M10 siRNA. Control and M10 siRNA-treated HeLa cell monolayers were infected with S. flexneri and subsequent plaque formation was enumerated. The numbers of plaques c ounted for Control cells and M10 siRNA cells was 92 and 68, respectively. P value is 0.0803.

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53 Figure 3-10. HeLa cells were ei ther transfected with non-targ eting siRNA (Ctrl siRNA) or M10 siRNA, infected with Shigella and analyzed using video microscopy. The average filopodial lengths are represente d. There was significant difference when Shigella -induced filopodial lengths from HeLa cells transfected with Ctrl siRNA were compared with those tr ansfected with M10 siRNA (14.73 m compared with 11.01 m, respectively. P value is 0.0005). Fo r cells transfected with Ctrl siRNA (black bar) and M10 siRNA (gray bar), n = 32 and 35, respectively. Data is representative of three independent experiments.

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54 CHAPTER 4 DISCUSSION M10 Recruitment to Shigella flexneri An important facet of pathogenesis for a ny pathogen is the ability to establish an infectious foothold in the host to propagate the infection. Shigella flexneri shares similarities with other intrac ellular pathogenic bacteria such as species of Listeria Rickettsia Mycobacterium, and Burkholderia in terms of the mechanism of actin-based intraand intercellular motility it has a dopted to spread an infection in the host (Carlsson and Brown, 2006). Due to the crucial role intercellular spread plays in successfully establishing infection, it is important to have a better understanding of the factors and m echanisms that govern Shigella induced filopodium formation. We have successfully shown that an unconventional myosin, M10, is recruited to intracellular motile Shigella undergoing filopodia formation and that its presence seems to promote the efficient forma tion of filopodia. M10 has previously been implicated in the process of filopodia formati on in eukaryotic cells (Berg and Cheney, 2002; Bohil et al. 2006) and this is the first tim e that this myosin has been implicated in the efficient formation of filopodia in an intr acellular bacterium. By eith er reducing endogenous levels of M10 in cells or expressing GFP-M10-HMM, we have shown that Shigella are less able to form filopodia of normal length when comp ared to unaltered ce lls (Figure 3-6 and 3-10). The inability of bacteria to form filopodia of normal length also seems to lead to a slowing down in the process of cell-to-cell spread as is eviden ced by the fewer number of plaques formed by Shigella on HeLa cell monolayers that have been treated with M10 siRNA compared to those formed on monolayers treated with c ontrol siRNA (Figure 3-9). Since cells expressing GFP-M10-HMM yielded Shigella -induced filopodia of lengths similar to cells in which endogenous M10 had be en knocked down, we supposed that the ability

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55 of M10 to aid in bacterial filopodia formation wa s due to one or more domains within the tail region. We found it difficult, however, to pinpoint the domain(s) that contained this property. Although Bohil and colleagues (2006) were ab le to show that deletion of the MyTH4-FERM region within the M10 tail was sufficient to nullify M10s ability to induce filopodia formation in eukaryotic cells, we found no indica tion that this was the case for Shigella -induced filopodia (Figure 3-7A and 3-7B). Furthermore, when we tried to inhibit the PH2 domain within the M10 tail region, we f ound no reduction in Shigella -induced filopodia lengths as compared to cells expressing the GFP-M10-HMM (Figure 3-8A C). Our results show that, in Shigella pathogenesis, only the complete absence of the en tire M10 molecule or simply the absence of the tail region hinders its ability to efficiently form filopodia and undergo efficient cell-to-cell spread. This seems to indicate that Shigella is not relying on the pres ence of any cargo that the M10 tail region might be delivering in order to fo rm filopodia, but rather requires the presence of the tail region perhaps to assist in proper lo calization and/or functiona lity of M10. It is interesting to note that in a recently published paper, Tokuo and colleagues (2007) demonstrated that only the motor function of the two-headed form of M10 is crucial in initiating filopodia formation in eukaryotic cells, wherea s, in regards to the process of Shigella pathogenesis, our findings seem to indicate that the presence of the PH domain-containing tail region is also required. It should be noted, however, that the filopodia observed in the Tokuo et al. study were only borne of preexisting lamellipodia in migrating cells and were relatively transient, quickly being retracted back into the lamellipodia. This is in some opposition to the study reported by Bohil et al. where they showed that the ability of M10 to induce filopodia formation was abolished when the MyTH4-FERM domains were deleted from the tail region.

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56 M10 Contribution During Shigella -induced Filopodium Formation In this study we have relayed one of the fi rst reports of an unconve ntional myosin being involved in efficient Shigella -induced filopodia formation and cell-to-cell spread. It has previously been reported that myosin motors are not invol ved nor required in the process of actin-based motility carried out by S. flexneri (Loisel et al ., 1999). Furthermore, there have been previous reports of myosins being recr uited to the phagocytic cup formed by Shigella upon entry into nonphagocytic host cells (C lerc and Sansonetti, 1987; Graf et al ., 2000), but their possible involvement in the process of filopodial formati on has not been intensively investigated. Some evidence pointing to the involve ment of host cell myosins in Shigella cell-to-cell spread was reported in 2000 by Rathman and colleagues. In their work, they found that the inhibition of MLCK, which is known to phosphorylate the light ch ain of myosin II, resulted in a marked decrease in Shigella s ability to disseminate in cultured Caco2 cell monolayers. Our study demonstrates that an unconventional myosin motor protein, M10, is recruited to motile intracellular Shigella as well as Shigella bound in filopodia (figure 3-2). We were also able to show that M10 recruitment was specific to Shigella and was not recruited to another intracellular pathogen that employs actin-based motility, L. monocytogenes (figure 3-1B). There is a similarly intriguing findi ng reported by Kolesnikova et al. (2006) of M10s co-involvement with actin in the budding release of a pa rticular virus. This group obser ved that the budd ing release of Marburgvirus (MARV) particles occurred almost exclusively at filopodia. When polymerization of actin in filopodia was inhibited, this resulted in a marked decreas e in total virus particles being released into the extracellular medium. Moreover, when M10 and/or Cdc42 were inhibited, the intracellular localization of a matrix protein known to play a key role in the release of MARV particles was concomitantly inhibited. In lieu of these findings the group went on to further theorize that perhaps by preferentially utilizing ho st cell filopodia at egress sites, the virus was

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57 capitalizing on these cellular projections clos e contact with neighbor ing cells and thereby enhancing the chances of successful invasion of adj acent cells. To date, this is the first finding of its kind to be reported in the field of viral pathogenesis and creates a striking parallel to our findings with M10 and S. flexneri s means of intercellular spread. At this point it isnt yet clear whether the recruitment of M10 to Shigella is mediated by a host cell factor or a bacterial component. There have only been a few studies examining Shigella s involvement in mediating efficient cell-tocell spread. One recent report cites the ability of the TTSS-secreted eff ector protein VirA to sever micr otubule filaments as an important means of facilitating optimal intraand intercellular spread (Yoshida et al ., 2006). Involvement of M10s Tail Region in Shigella -Induced Filopodium Formation The results we obtained from our experiment using Cos7 cells transfected with GFP-M10HMM that were then infected with Shigella indicated that the tail re gion of M10 contributes to some extent in aiding Shigella -induced filopodium formation (fi gure 3-6). For many myosins, the tail region is usually the area in which domains are found that confer cargo-binding specificity (Sellers, 2000). In the case of M10, many findings ha ve been reported that cite one or more of its tail domains responsible for conf erring some functionality to M10 in a broad array of cellular functions. Although no one specific functi on has been attributed to M10, it is mostly known for its role as a potent inducer of fil opodia on the surface of mammalian cells (Berg and Cheney, 2002; Bohil et al ., 2006). In 2002, Berg and Cheney were able to show that the head (motor) domain was responsible for properly loca lizing M10 to filopodial tips while Bohil and colleagues were later able to show that the MyTH4-FERM region was responsible for M10s ability to promote filopodia formation (Bohil et al ., 2006).

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58 Future Directions One of the main questions remaining about Shigella s ability to recruit host cell M10 is whether the recruitment is due to the presence of a bacterial or host cell factor. One possibility is that the absence of tropomyosin in Shigella -induced actin tails (Figure 3-3) simply makes actin available for M10 binding. However, it is unlik ely that this would be the sole reason if Shigella is in fact deliberately recrui ting M10 for a specific task. If Shigella were recruiting M10 by way of a bacterial factor, one candidate could be the IcsA protein. This protein stands out because its polarized distribution at one pole of motile bact eria is what enables directional motility in Shigella (Goldberg and Theriot, 1995) and because its reported distribution pattern closely resembles the pattern we see in M10 localization. Furthermore, this would be consistent with our finding that M10 recruitment is specific to Shigella and not Listeria another intracellular pathogen that utilizes actin-based motility via expression of the surface protein ActA (Gouin et al. 2005). Alternatively, if M10 were recruited to intracellular Shigella due to binding of a host cell factor, the absence of tropomyosin in actin ta ils could facilitate M10 motor activity while binding of host cell factors by the tail region could facilitate proper localization of the myosin molecule. This idea has been proposed previously (Tacon et al. 2004) and it is thought that the three tandem PH domains found in the M10 tail region are likely to be candidates for proper intracellular localization of M10. If this were the case, it may explain why we see truncated filopodia in cells expressing a M10-HMM cons truct, but not in cel ls expressing a M10FERM or a M10MyTH4FERM construct. The reason why fil opodia lengths are not truncated in the presence of compounds that hinder PH domain ac tivity, however, remains elusive. It may be possible that a sequence or set of sequences not associated with a defined domain exists in the region between the coiled-coil domain and the MyTH4 domain. It could be possible that the

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59 presence of sequences in this re gion facilitate binding of the ta il region to other proteins that could play a role in prop er intracellular M10 loca lization.

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68 Zhu, X-J., C-Z. Wang, P-G. Dai, Y. Xie, N-N. Song, Y. Liu, Q-S. Du, L. Mei, Y-Q. Ding, and W-C. Xiong. 2007. Myosin X regulates netrin receptors and functions in axonal pathfinding. Nature Cell Biol. 9:184-192. Zychlinsky, A., M.C. Prvost, and P.J. Sansonetti. 1992. Shigella flexneri induces apoptosis in infected macrophages. Nature. 358:167-169.

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69 BIOGRAPHICAL SKETCH Ellen Antoun Bishai was born on June 14, 1980 in Edmonton, Alberta, Canada. She and her family moved to Jersey City, NJ in 1983 an d finally settled in Tampa, FL in 1991. Ellen completed her undergraduate studies at the Univ ersity of South Florida in Tampa where she majored in biology and was an active member in th e viola section of the Un iversity Orchestra. Upon graduating in the Spring semester of 2003, she was enrolled in the Interdisciplinary Program in Biomedical Sciences (IDP) at the Univ ersity of Florida in the Fall semester of that year. There, she worked under the guidance of Dr. Frederick S outhwick in the completion of this dissertation. Ellen intends to continue her education by attendi ng the University of Pennsylvania, the University of Georgia, or th e University of Florida Veterinary School in pursuit of a doctor of vete rinary medicine degree.


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