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IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
LAURA DIANE ERRANTE
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
To my parents, with whom I realized how much I could accomplish if only I try,
and to my dearest Gator, who would never let me lose sight of my dreams.
Although it is now time to begin anew, I will never forget the people I have
met that have made the past six years a learning experience. Foremost I would
like to thank Dr. Gerry Shaw, my advisor, who showed me, through his example,
that although it may be difficult at times to do bench work, that this is why we
become scientists in the first place. In addition, I appreciate his patience,
especially in allowing me to develop my own sense of independence. I would like
to acknowledge my committee members, Dr. Barabara Battelle, Dr. Kevin
Anderson, and Dr. Daniel Purich, for their insight and support on my doctoral work.
I would like to credit Dr. Luttge for having the wisdom 5 years ago to let me know
that although I was having difficult times, I still had the ability to become a good
scientist. I only hope that the present and future graduate students in the
department will benefit from his wisdom as I have over these years. It is easiest
to remember to thank the people who have had direct input on your research;
however, I would like to give special thanks to Dr. Roger Heintz, my undergraduate
advisor at SUNY Plattsburgh, for instilling in me the basics of learning techniques,
especially column chromatography which has been essential to my dissertation
research, and for his encouragement to enter a field of study that can be
intimidating at times but is always fascinating and rewarding.
There are a number of graduate students and staff that have made an
indulible impression on me. In particular I will always treasure my friendships with
Paul Bao, Michele Davda, Bunnie Powell, and Margaret Tremwel. I would like to
acknowledge my family, especially my mom and brother, Wayne, who may not
have an understanding of what I have been doing the past six years but have
supported me just the same, no matter how infrequently they heard from me.
It has been a long and sometimes twisted road to reach this point, and I
am fortuitous to have found a true friend in Rob Friedman. Even when I thought
all was lost, his constant and unconditional support as well as critical assessment
of my research, enable me to envision the "light at the end of tunnel" and make
my dissertation a reality.
This work was supported by a NIH Predoctoral Training Fellowship
MH15737 through the Center for Neurobiological Sciences at the University of
Florida and NIH NS22695 (to Dr. Gerry Shaw). In addition, I would like to thank
the Center for Neurobiological Sciences at the University of Florida for providing
funds to attend scientific meetings.
TABLE OF CONTENTS
LIST OF TABLES ........................
LIST OF FIGURES .......................
LIST OF ABBREVIATIONS .................
. . . . .. xiv
1 INTRODUCTION AND BACKGROUND ................... 1
Cytoskeleton ..................................... 1
Intermediate Filament Family of Proteins ................ 2
Neurofilament Assembly ............................ 8
NF Phosphorylation ............................... 11
Interactions Between Neurofilaments and Microtubules ..... 18
Neurofilaments Interact with Other Cellular Components .... 21
Relationship of NFs to Axonal Transport and Caliber
NF Expression After Axotomy ................
Role of NFs in Disease States ................
Overview of Dissertation ....................
2 GENERAL METHODS .......................
Biochemical Techniques .......................... 36
Protein Assay ................................ 36
SDS Polyacrylamide Gels ........................ 37
Electroblotting of Proteins ........................ 38
Immuno-Blot Analysis ........................... 39
Protein Cleavage with Cyanogen Bromide ............ 40
Isolation of Neurofilament Proteins .................... 41
Crude Intermediate Filament Preparation ............. 41
Neurofilament Preparation ........................ 42
Purification of Individual Neurofilament Subunits ........ 43
Anatomical Localization Experiments .............
Dorsal Root Ganglion Cell Cultures ............
Pheochromocytoma Cells (PC12 Cells) .........
Immunofluorescent Studies on Fresh Frozen Tissue
Immunocytochemistry on Formaldehyde Fixed Tissue
3 THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE
FILAMENT BINDING PROTEIN, IN THE ADULT RAT
CENTRAL NERVOUS SYSTEM ..................
M ethods .................................
Immunoblot Studies ........................
Comparison of Plectin to IFAP-300 .............
Localization Studies ........................
N otes ...................................
4 CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM .......
M ethods .................................
Immunoblot Studies ........................
Co-localization in Cultured Cells ...............
Co-localization Studies Along the Rat Neural Axis .
Effects of Peripheral Nerve Axotomy .............
N otes ................................... .
5 IDENTIFICATION OF NF BINDING PROTEINS .
M ethods ............................
Pig Spinal Cord Cytosolic Preparation .....
Affinity Column Preparation
Binding of Cytosolic Proteins to Affinity Columns ..
Production of Polyclonal and Monoclonal Antibodies
. .. 44
. .. 46
6 CHARACTERIZATION OF GAPDH BINDING TO NFs ..
M ethods ..............................
Co-sedimentation Experiments ............
Immunofluorescence Studies ..............
Co-sedimentation Experiments ............
Immunofluorescence Studies in Cultured Cells .
7 OVERALL DISCUSSION ....................
General Considerations ...................
Criteria for Classifying IF Binding Proteins .....
Relationship between Plectin and NF Proteins .
The Role of GAPDH in the Nervous System ....
Future Direction ........................
BIOGRAPHICAL SKETCH ..........................
. ... 165
. ... 166
. ... 168
LIST OF TABLES
Mammalian intermediate filament protein family based
on the classification scheme of Steinert and Roop (1988) .... 2
Protein kinases which phosphorylate neurofilaments in vitro 14
Interactions of neurofilaments with other cellular components 22
The distribution of plectin in different cell types of the adult
rat central nervous system .......................... 76
Summary of the distribution of plectin in different cell types
of the adult rat nervous system with respect to
intermediate filament proteins ....................... 111
Relative molecular weight (kD) of candidate proteins which
bound to the five different types of NF affinity columns .... 123
Amino acid composition for candidate neurofilament binding
proteins with the data represented as nanomole percent .... 127
Results from the FINDER program to identity NFL-38 ..... 131
Comparison of NFL-38 to GAPDH with data represented
as nanomole percent for each amino acid .............. 132
LIST OF FIGURES
Schematic of neural intermediate filament protein sequences 5
Identification of a 300 kD protein in bovine and rat crude
IF spinal cord preparations ......................... 55
Comparison of amino acid composition of plectin and
IFAP-300 proteins ............................... 58
Plectin immunoreactivity (ID8) of astrocytes in white and
gray matter of the telencephalon and diencephalon ....... 61
Plectin immunoreactivity (ID8) in the rostral hypothalamus
surrounding the third ventricle (3V) as visualized with DAB .. 64
Plectin immunoreactivity (IA2 and ID8) in the cerebellum ... 66
Plectin immunoreactivity (ID8) in the caudal brainstem ..... 69
Plectin immunoreactivity (ID8) in the cervical spinal cord .... 72
Plectin immunoreactivity (p21) in choroid plexus and
blood vessels .................................. 74
Characterization of peripherin polyclonal and monoclonal
antibodies ..................................... 84
Co-localization of plectin (p21) and vimentin (V9) in
non-neuronal cells from DRG cultures ................ 87
Co-localization of plectin (ID8) and NF subunits in
DRG cells and PC12 cells ......................... 89
Co-localization experiment with antibodies to plectin
(p21) (A,C,E,G) and vimentin (V9) (B,D,F,H) ............ 93
Co-localization experiment with antibodies to plectin
(A,C,E) and GFAP (B,D,F) ........................ 95
Co-localization experiment with antibodies to plectin
(ID8) (A,C,E) and NFs polyclonall) (B,D,F) .............. 99
Co-localization experiment with antibodies to plectin
(IA2) (A,C) and peripherin (R19) (B,D) ................ 101
Effects of a unilateral facial nerve axotomy on plectin
Figure 4-9 Co-localization experiment with plectin (IA2) (A,C)
and NF-H (R14) (B,D) after a unilateral facial nerve
axotom y .....................................
Figure 4-10 Co-localization experiment with plectin(IA2) (A,C)
and peripherin (R20) (B,D) after a unilateral facial
nerve axotomy .................................
Figure 5-1 Outline of the method used for identifying candidate
NF binding proteins .............................
Figure 5-2 Comparison of candidate 38 kD NF binding protein
to rod domain of IF proteins .......................
Figure 5-3 Comparison of amino acid composition data between
NFL-38 and IF-38, and to that of the average amino
acid composition in Genbank ......................
Figure 5-4 Comparison of CNBr cleavage fragments of NFL-38
and GAPDH ..................................
Figure 5-5 Immunoblot analysis using antibodies to GAPDH to
determine if the 38 kD proteins binding to various NF
affinity columns is GAPDH ........................
Figure 5-6 Graphical representation of the amino acid composition
data for NFL-16, and possible matches as determined
by the FINDER program ..........................
Figure 5-7 Graphical representation of the amino acid composition
data for NFL-62, and to that of the average amino acid
composition in Genbank ..........................
Figure 6-1 Co-sedimentation experiment with GAPDH and NF subunits
Figure 6-2 Co-sedimentation of GAPDH and NF-L with different
concentrations of GAPDH .........................
Figure 6-3 Co-sedimentation of GAPDH and NF-L with different
concentrations of sodium chloride ...................
Figure 6-4 Composite of GAPDH antibody labelling (ID4) of a DRG
cultured neuron ................................
Figure 6-5 Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A,C) and NF-H (R14) (B,D) .
.... ..... .... ... .. .. .. .. .. 104
Co-localization experiment in differentiated PC12
cell cultures with antibodies to GAPDH (ID4) (A,B)
and NF-M (R9) (C) ............................... 160
Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A) and actin polyclonall) (B) ..
LIST OF ABBREVIATIONS
ALA or A
ARG or R
ASN or N
ASP or D
ASX or Z
CYS or C
GLN or Q
GLU or E
GLX or B
GLY or G
HIS or H
ILE or I
LEU or L
LYS or K
MET or M
PHE or F
PRO or P
SER or S
THR or T
TRP or W
TYR or Y
- aspartic acid
- asparagine and/or
- glutamic acid
- glutamine and/or g
BBB blood-brain barrier
BSA bovine serum albumin
CBB Coomassie brilliant blue R-250
CNBr cyanogen bromide
central nervous system
dorsal root ganglion
EDTA ethylenediamine tetraacetic acid
FBS fetal bovine serum
GAPDH glyceraldehyde-3-phosphate dehydrogenase
GFAP glial fibrillary acidic protein
GST glutathione S-transferase
IF intermediate filament
IFAP intermediate filament associated protein
MES 2-(4-morpholino)-ethanesulfonic acid
NF-H neurofilament high molecular weight subunit
NF-M neurofilament middle molecular weight subunit
NF-L neurofilament low molecular weight subunit
TBS tris buffered saline
PBS phosphate buffered saline
PMSF phenylmethyl-sulfonyl fluoride
PNS peripheral nervous system
PVDF polyvinylidine difluoride
SDS sodium dodecyl sulfate
TAME Na-p-toluenesulfonyl-L-arginine methyl ester
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
IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
LAURA DIANE ERRANTE
Chairperson: Gerard P. J. Shaw
Major Department: Neuroscience
Neurofilaments (NFs), microfilaments (MFs) and microtubules (MTs) are the
major structural proteins that form the neuronal cytoskeleton. Much more is
understood about the function of MFs and MTs than is known for NFs. This
dissertation research characterized, plectin, a known intermediate filament
associated protein (IFAP) in the nervous system, and searched for candidate NF
binding proteins in order to examine possible roles for NFs.
Plectin distribution was examined throughout the rat CNS. Plectin was
localized to non-neuronal cells with particularly strong immunoreactivity in cells
forming ventricular and pia barriers. In addition, plectin immunoreactivity was
observed in select motoneurons. Double-label studies with plectin and IF proteins
demonstrated that plectin's distribution most closely resembled that for vimentin;
however, the staining patterns were not mutually inclusive. In select motoneurons,
plectin colocalized with the neural IF proteins, NF triplet proteins and peripherin.
Since plectin and NFs co-exist in the same cell, the previously described in vitro
plectin/NF interactions may be functionally important.
The second part of the dissertation project involved identifying a 38 kD
protein which bound to NF affinity columns. Using a computer program, FINDER,
and biochemical analysis, this protein was identified as glyceraldehyde-3-
phosphate dehydrogenase (GAPDH). The strength of binding between GAPDH
and NF-L subunit was shown to be relatively weak with a dramatic decrease in the
amount of protein co-pelleting at 0.05 M sodium chloride. The possible in vivo
interaction between NFs and GAPDH was examined using fluorescent light
microscopy which showed that GAPDH was localized throughout the cell body and
processes of dorsal root ganglion cells in culture and differentiated PC12 cells.
Although GAPDH immunoreactivity colocalized in part with NF antibody labelling,
the immunofluorescent labelling patterns were distinct.
A number of proteins were identified which bound with varying specificity to
NFs in vitro and have a potential of interacting with NFs in vivo. Finding that NFs
may interact, in vivo, in some neurons with plectin and GAPDH suggests that
certain NF proteins may act as important structural elements during neuronal
injury, and as docking substrates for the localization of glycolytic enzymes.
INTRODUCTION AND BACKGROUND
The cytoskeleton is a three-dimensional network of fibrous proteins found
throughout the cytoplasm. The neuronal cytoskeleton is composed of microtubules
(MTs), microfilaments (MFs) and neurofilaments (NFs). MTs and MFs have been
well characterized in both neuronal and non-neuronal cells, and both have been
shown to have a variety of roles in cell structure and motility. In contrast, very little
is understood about the functional role of NFs (Fliegner and Liem, 1991; Shaw,
1991). NFs are primarily thought of as static structures ensuring mechanical
stability to the neuron and governing the diameter of the axon (Hoffman et al.,
1984). However, changes in phosphorylation state of NFs during development and
regeneration indicate that NFs may play a more active role in neuronal function.
This chapter will describe pertinent information concerning NF structure, assembly,
phosphorylation, interactions with other proteins, and NFs involvement in axonal
transport and caliber. The last part of this chapter will discuss the effects on NF
proteins of axotomy and neuronal diseases which may indicate possible functions
for these neural intermediate filaments.
Intermediate Filament Family of Proteins
NFs belong to a family of intermediate filament (IF) proteins which are 10
nm diameter. The IF family is currently grouped into 6 classes of proteins based
on protein sequence similarity and intron placement (Table 1-1).
Table 1-1. Mammalian intermediate filament protein family based on the
classification scheme of Steinert and Roop (1988).
Class Name MW (kD) Cellular Location
I Keratin (acidic) 40-60 epithelial cells and epidermal
derivatives (eg. nails and hair)
II Keratin (basic) 50-70 "
III Desmin 52 muscle cells
Vimentin 57 cells of mesenchymal origin
GFAP* 51 astrocytes; some schwann cells
Peripherin 57 neurons
IV NF-triplet proteins
NF-H1 115 neurons
NF-M' 95 "
NF-L* 60 "
a-Internexin 66 "
V Nuclear Lamins 60-70 nuclear lamina of all cells
IV Nestin 200 neural epithelial stem cells
*GFAP: glial fibrillary acidic protein; 1NF-H: NF high molecular weight protein;
ONF-M: NF middle molecular weight protein; "NF-L: NF low molecular weight protein
A number of IF proteins are localized to neurons. The classical NFs, NF
triplet proteins, are found in varying concentration in most nerve cells. (Liem et al.,
1978; Yen and Fields, 1981; Shaw et al., 1981). In addition to the NF triplet
protein, a-internexin and peripherin are localized to a more select set of neurons
and have been shown to co-localize with NF-triplet protein (Kaplan et al., 1990;
Parysek et al., 1991). a-lnternexin is distributed predominantly in the central
nervous system (CNS) (Pachter and Liem, 1985; Chiu et al., 1989). Peripherin is
localized mainly in the peripheral nervous system (PNS) (Portier et al., 1984a,
1984b), but is found also in select fiber tracks and perikarya of neurons throughout
the CNS (Parysek and Goldman, 1988; Brody et al., 1989). Although vimentin is
primarily thought to associate with non-neuronal cells, it is localized in developing
neurons and is expressed in mature neurons in the olfactory epithelium (Schwob
et al., 1986) and adult retina (Draeger, 1983). Nestin, a newly described protein
assigned to class VI, is widely expressed in neuroepithelial stem cells which are
the precursors of CNS neurons and glia (Hockfield and McKay, 1985; Frederiksen
and McKay, 1988; Lendahl et al., 1990).
IFs, in general, have a fundamental structure comprised of 3 domains: (1)
amino-terminal head; (2) central a-helical rod domain; and (3) variable carboxyl-
terminal tail (Figure 1.1). The amino-terminal head domain is typically a short
globular peptide sequence of 5-7 kD containing numerous basic amino acids
(Geisler et al.,1982). However, the amino-terminal domain of nestin is only 11
amino acids long and is slightly acidic (Lendahl et al., 1990). The central rod
domain (39 kD) contains a highly conserved heptad repeat of hydrophobic amino
acids that allows for the formation of an a-helical coiled-coil structure (Geisler et
al., 1982). The distinct primary amino acid sequence of the rod domain is
proposed to be the driving force of IF protein self-assembly (Steinert and Roop,
E: o.. |z
1988). One unifying feature of all IFs is a highly conserved epitope at the end of
the rod domain (coil II) which is recognized by the anti-intermediate filament
antibody (a-IFA) (Pruss et al., 1981). Most IF subunits, such as vimentin, have a
5-10 kD globular tail sequence at the extreme C-terminus of .the molecule.
However, the class IV and V IF proteins differ from other IFs in that they have long
carboxyl-terminal extensions which contain several distinct types of unusual amino
The NF carboxyl-terminal tails appear to have very little a-helical or B-sheet
structure and are thus thought to consist of largely random coils (Geisler et al.,
1985b). The NF tail domain can be divided into 4 regions: (1) tail A; (2) glutamic
rich segment (E segment); (3) lysine-serine-proline repeat segment (KSP
segment); and (4) lysine and glutamic rich segment (KE or KEP segment). It has
yet to be determined if these sequence specific regions are actually functional
domains. In contrast to the other IF subunits, nestin's carboxyl-terminal tail domain
has a distinct repeat segment (KEQP) which is repeated 35 times (Lendahl et al.,
1990). The abundance of glutamic acid residues in the carboxyl-terminal of nestin
is the only similarity with the carboxyl-terminal of class IV IF proteins (Fliegner and
The latter two segments of NF-M and NF-H carboxyl terminal tails (KSP and
KE or KEP) are thought to correspond to the rodlets protruding from the core
filament seen in ultra-structural studies (Hisanaga and Hirokawa, 1988). The
sequence motif of K-SP or K--SP is also present in microtubule associated proteins
tau and MAP2 (Shaw, 1989; Shaw, 1991). The K-SP and K--SP sites in pig NF-M
and K--SP site in rat NF-M are phosphorylation sites in vivo (Geisler et al., 1987;
Xu et al., 1989). At the extreme terminus of NF-M is a highly conserved region
known as the KE segment which is rich in lysine and glutamic acid. This highly
charged segment may be important in interactions with other neuronal components
since it is highly conserved and a closely related sequence is also present in the
extreme C-terminus of a.-internexin (Shaw, 1991).
NF-H is somewhat similar to NF-M but has a distinct tail domain. In
contrast to NF-M, NF-H has a KSP segment that contains far more repeats of the
KSP motif. In rat, this KSP motif is repeated 60 times. In addition, the KSP repeat
sequences in NF-H are distinct from those found in NF-M (Shaw, 1991). Instead
of a KE region at the C-terminus, NF-H has a segment rich in lysine, glutamic acid
and proline (KEP) which is evolutionarily more loosely conserved (Shaw, 1991).
Recently, the different tail segments of NF-M and NF-H have been
dissected out molecularly and expressed as fusion proteins (Harris, et al., 1991).
These specific segments of the high MW NF tail regions can be used to examine
the functional significance NF through examining their interactions with NFs, other
neuronal components and by using these defined protein segments as substrates
for protein kinase assays.
The way in which individual NF subunits interact and assemble into NFs is
not clearly understood. NF-L appears to be the core structure of assembled 10 nm
filaments since urea solubilized NF-L monomers self assemble into homopolymeric
structures when urea is removed (Geisler and Weber, 1981). NF-M and NF-H
appear to be incapable of forming long 10 nm filaments, however, when NF-L is
present, both NF-M and NF-H are readily incorporated into the 10 nm filament
(Geisler and Weber, 1981; Liem and Hutchison, 1982). Antibody studies (Willard
and Simon, 1981; Hirokawa et al., 1984) and rotary shadowing experiments
(Hisanaga and Hirokawa, 1988) support the idea that NF-L is the central core of
the filament, and NF-M and NF-H are incorporated into this core by their rod
domains where the carboxyl-terminal tail domains of NF-M and NF-H protrude from
the central core of the filament. Recently, rotary shadowing experiments with
antibodies to the tail domain of NF-M and NF-H confirm that the tail regions of NF-
M and NF-H correspond to the protrusions seen ultrastructurally with NFs (Mulligan
et al., 1991). A basic question which remains is how individual IF subunits, in
general, interact to form 10 nm filaments.
The majority of research on IF assembly has focused on class III IFs,
desmin and vimentin, which can form homopolymers in vivo. When IFs form
dimers it is believed that the hydrophobic portions of the a-helical coil regions of
two polypeptide chains interact in a parallel fashion and form a coiled-coil structure
(Parry et al., 1982). In addition to satisfying hydrophobic considerations, the coil-
coil type structure allows for favorable charge interactions along the rod domain.
The dimers are then thought to form four chain tetramers either in a parallel (Ip,
1988; Hisanaga et al., 1990), anti-parallel (Geisler et al., 1985a) or staggered anti-
parallel manner (Stewart et al., 1989). The differences in tetrameric formation may
be related to the different IF subunits used or the way in which tetramers interact
with the intact filament network (Steinert and Roop, 1988). Higher oligomers are
then believed to form by a staggering of the tetramers which then results in the
formation of intermediate filaments (Coulombe and Fuchs, 1990; Ip et al., 1985).
Eight tetramers are thought to comprise a single 10 nm filament.
Recently the assembly process of NFs was studied using low angle rotary
shadowing. These studies showed that the basic building block of IF proteins, NF-
L and GFAP, were an eight chain structure formed from two parallel tetramers
(Hisanaga et al., 1990), where four octamers were proposed to comprise a single
10 nm filament. Differences were also found in the way in which the filaments
were reassembled. Hisanaga et al. (1990) showed that NF-L can assemble into
either four chain or eight chain complexes depending on the pH of a low ionic
strength buffer. In preceding experiments, alkaline buffer (pH 8.5) produced
tetramers (Huiatt et al., 1980; Geisler and Weber, 1982; Ip et al.,1985; Hisanaga
et al., 1990); whereas low salt buffer (pH 6.8) favored the formation of octamers
(Hisanaga et al., 1990).
Hisanaga and Hirokawa (1990a) also examined how NFs assemble end to
end to form long 10nm filaments. They found that in the polymerization of NF-L
monomers, sodium ions were important for end-t- end association, whereas
magnesium ions were involved in stabilizing lateral associations. When NFs were
reassembled using NF-L, NF-M and NF-H, the assembly conditions remained
unchanged; however, there was a structural change. With addition of either NF-M
or NF-H, rod-like projections were seen emanating from the central core of the
filament similar to those previously seen using native NFs (Hisanaga and
The domains of individual NF subunits have been examined to determine
which portions of NF sequence are necessary for assembly into 10 nm filaments.
Genetic deletion of the amino terminal head domain and carboxyl terminal tail
domain of the mouse NF-L gene showed that deletions larger than 30% from the
head domain and 90% from the tail domain prevented incorporation of these
proteins into the intermediate filament network (Gill et al., 1990). In contrast, when
deletions were made in NF-M gene, up to 70% of the head domain and 90% of the
tail domain could be missing and NF-M would still be incorporated into filaments
(Wong and Cleveland, 1990). However, deletions into either amino- or carboxyl-
terminal region of the a-helical rod domain of NF-M prevented assembly of this
gene product into the filament network. Therefore, the intact rod domain was
determined to be critical for NF assembly.
Phosphorylation of NF subunits were found to effect filament assembly.
Gonda et al. (1990) demonstrated that protein kinase C phosphorylated a number
of serine residues in the N-terminal head domain of NF-L and that an increase in
phosphate incorporation decreased the rate of polymerization of NF-L monomer
into filaments. In addition, cAMP dependent protein kinase was shown to
phosphorylate NF-L and was found to inhibit assembly of NF-L monomers into
filaments at phosphorylation levels of 1 mol/mol of protein (Nakamura et al., 1990).
Finally, phosphorylation of NF-L filaments resulted in a slow disassembly which
took up to 6 hours. Nakamura and co-workers proposed that controlled
phosphorylation may modulate the dynamic equilibrium between assembly and
disassembly in vivo.
The NF triplet proteins are phosphorylated in vivo and the relative amount
of phosphorylation in rat spinal cord was determined by one group to be 3, 6 and
13 moles of phosphate/mole of NF-L, NF-M and NF-H, respectively (Julien and
Mushynski, 1981; Julien and Mushynski, 1982; Xu et al., 1990). It is important to
note that the relative amount of phosphate associated with NFs is variable and
dependent on a number of factors including species, protein preparations, and area
of the nervous system examined. What is generally known is that NF-H appears
to contain the most phosphate per mole of polypeptide followed by NF-M then NF-
L. In the case of NF-H and NF-L the phosphate is associated with serine residues,
whereas, NF-M has phosphorylation sites on both serine and threonine residues
in the rat (Julien and Mushynski, 1982).
Phosphorylation sites on NF triplet proteins are localized to particular
regions on the individual NF subunits. In the case of rat NF-L, phosphorylation
sites are found in both the amino head and carboxyl tail domains (Sihag and
Nixon, 1989) with the major phosphorylation site located in the middle of carboxyl-
terminal tail domain (Xu et al. (1990). In addition, in vitro phosphorylation
experiments with NF-L and protein kinase C show that there are 3 phosphorylation
sites on serine residues in NF-L amino-head domain (Gonda et al., 1990). A
detailed study of NF-M phosphorylation sites of rat neurons in tissue culture
demonstrated that there are six sites in NF-M carboxyl-terminus, four of which
have the KSP motif, one with the K--SP motif, and one motif with a serine residue
followed by glutamic acids (Xu et al., 1989). For NF-H the only known
phosphorylation sites are in the multiple KSP repeats, though it is likely that other
phosphorylation sites exist in other regions of this large molecule (Geisler et al.,
1987: Lee et al.,1988).
Three major questions still remain with respect to NF phosphorylation: (1)
what kinase activities are phosphorylating these different regions of NF subunits
in vivo; (2) are these kinases unique to NFs and/or are they previously
characterized protein kinases; and (3) what is the function of NF phosphorylation?
A number of laboratories have examined NF phosphorylation (Table 1.2). The
research with known kinases has focused primarily on NF-L subunit; whereas, NF-
M and NF-H have been utilized to search for novel protein kinase activities
responsible for phosphorylating the KSP repeat motif of NFs.
Three classical protein kinases were shown to phosphorylate NFs in vitro
to various degrees (Table 1-2): (1) calcium/calmodulin protein kinase II (CaM
kinase II) (Vallano et al., 1985); (2) cyclic AMP-dependent protein kinase (protein
kinase A) (Tanaka, 1984; Dosemeci and Pant, 1992); and (3) protein kinase C
(Sihag et al., 1988). In vivo phosphorylation experiments examining NF-L and NF-
M demonstrated that most of the fragments that are phosphorylated with one of the
known protein kinases are located in the amino terminal head domain (Sihag and
Nixon, 1989, 1990).
The search for the protein kinase which phosphorylates the KSP repeat
motif in NF-M and NF-H has focused on characterizing the protein kinase activities
which bind strongly to NFs during crude purification procedures (Table 1-2). These
kinase activities are extracted from NF preparations using high salt buffer and have
been shown to contain a number of protein kinases: (1) CaM kinase II; (2)
nucleotide-dependent kinase; (3) calcium-phosphatidylserine-diglyceride-dependent
kinase; and (4) a regulator independent kinase (Dosemeci et al, 1990). The
regulator independent kinase phosphorylated all three NF subunits as well as a-
casein where the level of phosphate incorporated per mole of protein was in the
following order: a-casein > NF-M > NF-H = NF-L. This kinase activity was shown
to be related to casein kinase I based on: (1) similarities in specificity of protein
kinase inhibitors; (2) the ability of casein kinase I to phosphorylate NFs in a similar
manner to the NF-associated kinase; and (3) the molecular weight range for casein
Table 1-2. Protein kinases which phosphorylate neurofilaments in vitro.
Protein Kinase Substrate MW* (kD) Reference
Protein Kinase C
Casein Kinase I
NF-H, -M, -L
NF-H, -M, -L
NF-H, -M, -L
NF-H, -M, -L
NF-H, -M, -L
NF-H, -M, -L
NF-H, -M, -L
Tanaka et al., 1984
Dosemeci and Pant, 1992
Vallano et al., 1985
Sihag et al., 1988
Floyd et al., 1991
Hisanaga et al., 1991
Lew et al., 1992
Toru-Delbauffe & Pierre, 1983
Toru-Delbauffe et al., 1986
Wible et al., 1989
Dosemeci et al., 1990
Floyd et al., 1991
* molecular weight expressed in kiloDaltons
' proline directed kinase
kinase I (Hathaway and Traugh, 1982) was comparable to that of NF-associated
kinase (Floyd et al., 1991).
Using the salt extraction technique, other laboratories have isolated
additional NF protein kinase activities, where their molecular weight and substrate
specificity suggest that these NF kinase are distinct from each other. Toru-
Delbauffe and Pierre (1983) were able to recover two endogenous protein kinase
activities, one which phosphorylated NFs, and the other which phosphorylate
casein and was identified as casein kinase I. The NF specific protein kinase was
purified by phosphocellulose chromatography and resulted in major band at 40 kD
and a minor band at 200 kD on SDS polyacrylamide gels, and appeared to be co-
factor independent (Toru-Delbauffe et al., 1986). Wible et al. (1989) partially
purified a 67 kD doublet on SDS polyacrylamide gels and, in contrast to the
previous kinases, their kinase showed more substrate specificity with a preference
for partially dephosphorylated NF-H.
Even though some of the kinases had a preference for the
dephosphorylated form of NF-H, phosphorylation by these various NF kinase
activities did not result in the expected molecular weight shift if the
dephosphorylated NF-H was being phosphorylated in the KSP repeat sequence.
Previous work has demonstrated that the two high molecular weight NF subunits,
NF-H and NF-M, mobility on SDS polyacrylamide gels is greatly retarded due to
the large number of phosphates in the carboxyl-terminal tail domain. Hisanaga
and co-workers (1991) demonstrated that the cdc kinase from starfish, p34cdc2,
phosphorylated both NF-H and NF-M in vitro. Furthermore, after phosphorylation
of enzymatically dephosphorylated NF-H, NF-H migration on SDS polyacrylamide
gels was similar to that observed for the native condition where NF-H is heavily
phosphorylated. Similar results were also obtained with a cdc2 kinase,
p34Cd2/p58CycinA, isolated from FM3A mouse mammary carcinoma cells (Guan et
al., 1992). These findings appear to be significant since cdc kinases are proline
directed kinases which specifically phosphorylate the serine residue on the
sequence S/T-P making the repeat KSP related sequences in NFs likely
substrates. However, cdc kinases are associated with the regulation of cell cycle
and have not been localized to neuronal cells which leads one to question the
relevance of in vitro phosphorylation of NF by cdc2 kinases.
Recently, a proline directed protein kinase was isolated from bovine brain
that consisted of two subunits, 33 kD and 25 kD (Lew et al., 1992a). This kinase
was shown to phosphorylate NFs, where the 33 kD subunit displayed a high
sequence homology to p34cdc2 kinase (Lew et al., 1992b). Independent, cloning
techniques identified a neuronal cdc2-like kinase (nclk) identical to that isolated by
Lew et al. (1992b) which had a 58% homology to mouse p34cd2 kinase (Hellmich
et al., 1992). In situ hybridization experiments demonstrated that unlike p34cdc2
kinase, nclk was expressed in high levels in terminally differentiated neurons
(Hellmich et al., 1992).
Complexities in studying NF phosphorylation seems to revolve around the
phosphorylation state of the NF subunits. Most of these studies use NFs which
are already phosphorylated in vivo. When NFs are used in this form, the in vivo
phosphorylating sites for a specific kinase may already be phosphorylated. In
addition, the phosphorylation state of NFs may effect the activity of a NF kinase
(Wible et al., 1989). Another problem with complete enzymatic dephosphorylation
is that some phosphate groups are more susceptible to a phosphatase than others,
thus all of the phosphates may not be removed. And finally, the number of
potential phosphorylation sites that exist in NF subunits make it difficult to
determine where a specific NF-kinase is acting.
Since NFs are phosphoproteins and seemingly contain a number of
potential sites for phosphorylation, the next obvious question is what is the role of
NF phosphorylation. The distribution of phosphorylated NFs differs within the
neuron. NFs are heavily phosphorylated in the axon, whereas the dendrites and
cell body appear to contain little or no phosphorylated NFs (Sternberger and
Sternberger, 1983). Even when considering that NF phosphorylation may be
involved in calcium buffering during nerve excitation and regulation of NF
assembly/disassembly, the functional significance of the phosphorylation
differences within the neuron is still not understood.
Ultrastructural difference between NFs found in axons and dendrites was
apparent with deep-etch freeze fracture technique (Hirokawa, 1982; Hirokawa et
al., 1984). NFs appeared to be spaced farther apart in axons then they were in
dendrites. These studies also revealed what appeared to be extensive cross-links
between the NFs in axons which were not seen with other intermediate filaments.
Therefore, it was hypothesized that the carboxyl-tail domains of NF-M and NF-H
were mediating this cross-linking, and that the length of the cross-links were
regulated by the amount of phosphate incorporation in the KSP segments.
However, no effect on the structure or length of NF tail projections were observed
upon enzymatic dephosphorylation which removed around 90% of the phosphate
groups (Hisanaga and Hirokawa, 1989).
In conclusion, there appears to be a diversity of protein kinase activities
capable of phosphorylating NFs. At least some of these phosphorylation events
are under tight spatial control in the neuron. Since NF subunits contain numerous
potential phosphorylation sites, we can expect that different phosphorylation events
will have different functional significance in the neuron.
Interactions Between Neurofilaments and Microtubules
Indirect findings suggest an interaction between NFs and MTs.
Anatomically, NFs and MTs run parallel to each other in the longitudinal direction
in axons and dendrites, and there appears to be morphological cross-bridges
between these two structures (Wuerker and Palay, 1969; Burton and Fernandez,
1973; Rice et al., 1980; Hirokawa, 1982; Hirokawa et al., 1984). In addition,
biochemical evidence shows that: (1) NFs co-purify with MTs prepared by the
polymerization-depolymerization method (Berkowitz et al., 1977); and (2) both NFs
and MTs travel in the slowest component of axonal transport (Hoffman and Lasek,
More direct evidence demonstrating NF-MT interactions showed that NFs
inhibit tubulin polymerization. This MT assembly inhibition was found to be the
result of NFs binding to high molecular weight microtubule associated proteins
(MAPs) which are known to catalyze MT assembly (Leterrier et al., 1982). To
further examine this interaction, Aamodt and Williams (1984) compared the ability
of NF isolated from brain (NF-Brain) with that of NFs isolated from spinal cord (NF-
SC) to form viscous complexes with MTs. They demonstrated that only NF-Brain
were able to form viscous complexes with MTs. They hypothesized that NF-SC
did not have MAPs bound to them; a suggestion supported by the finding that
when purified MAPs were added to NF-SC and MTs, a viscous solution formed.
Flynn et al. (1987) went on to show that NFs bound to the carboxyl-terminal
thrombin fragment of MAP2 (28 kD) which also is the MT binding site. Solid-phase
binding techniques show that NF-L was the only NF protein to bind to MAP2
(Heimann et al., 1985). The 28 kD fragment of MAP2 was also found to bind to
purified NF-L (Flynn et al., 1987).
To examine a possible in vivo interaction of MAP2 binding to both NF and
MT network, B,B'-iminodipropionitrile was used to segregate the two cytoskeletal
networks in peripheral nerves. Two different monoclonal antibodies to MAP2
demonstrated that one antibody to MAP2 co-localized with the NF network while
the other co-localized with MT network, suggesting that MAP2 is acting as a
crosslinker between these two fibrous networks (Papasozomenos et al., 1985).
In addition to MAP2 binding to NF-L, another MT associated protein, tau,
bound to reassembled NF-L. Competition studies with MAP2 and tau for binding
to NF-L demonstrated that these proteins bound to different sites on NF-L. The
in vivo significance is in question since the affinity of either MAP2 or tau was
decreased when NF-M and NF-H were present in the filamentous form as normally
is the case in vivo (Miyata et al., 1986).
Hisanaga and Hirokawa (1990b) examined the ability of NF subunits to bind
directly to polymerized MTs, and showed that dephosphorylated NF-H bound to
polymerized MTs in vitro. Further work by Hisanaga and co-workers (1993)
suggested that the binding of dephosphorylated NF-H to MTs was not dependent
on the degree of dephosphorylation but on the removal of a particular group of
phosphates. This was demonstrated by using two different phosphatases; alkaline
phosphatase and acid phosphatase. Partially dephosphorylated NF-H co-
precipitated with MTs in a similar manner even though alkaline phosphatase
removed 46 of 51 phosphates on NF-H whereas only 19 phosphates were
removed with acid phosphatase. In addition, this binding of dephosphorylated NF-
H to MTs is thought to occur at the carboxyl terminal tail region of NF-H.
NFs were shown to promote tubulin assembly in vitro (Minami et al., 1982);
however, NF preparations used in these initial studies were contaminated with
MAPs so that the MAPs may have been a factor promoting tubulin assembly. To
clarify these results, Minami and Sakai (1983) demonstrated that highly purified NF
preparations retained their ability to promote MT assembly as measured by a
gelation assay. Adding purified NF subunits to the assay demonstrated that NF-H
was the subunit responsible for promoting tubulin assembly. NF-M was able to
induce some MT assembly whereas NF-L had no effect on MT assembly.
Negative stain images of MT assembled in the presence or absence of NFs
showed that there was an increase in MT assembly in the presence of NFs. When
these structures were examined under the electron microscope, there appeared
to be no morphological differences between the assembled microtubules. In the
electron micrographs, the NFs appeared to make a lateral association with the MTs
which resembled a T shaped structure.
The role of NF binding to MTs is not known but it is believed that the tail
regions of the high molecular weight NF proteins are involved in some cross-linking
function. MAP2 is localized in dendrites and may be mediating some of the
interactions between NFs and MTs; whereas, tau is localized in axons where it
may be performing a similar function.
Neurofilaments Interact with Other Cellular Components
The interactions of NFs with other components of the neuron is not as well
studied as those interactions between NFs and MTs (Table 1-3). The majority of
proteins that will be discussed below bind primarily to NF-L.
Brain spectrin (fodrin) is closely associated with the plasma membrane
throughout the neuron (Levine and Willard, 1981) and is thought to play an active
role in stabilizing the membrane as well as immobilizing certain membrane proteins
c- 0 0
( > a
. oE .c.
U. U.- .LL
(Hirokawa, 1989). Frappier and co-workers (1987) demonstrated that brain
spectrin associates with NF-L and GFAP reversibly and in a concentration
dependent manner using in vitro binding assays. Cleavage with BNPS-skatole
reagent demonstrated that the binding site for brain spectrin was localized to the
40 kD N-terminal fragment which includes the head domain of brain spectrin and
the proximal portion of the rod domain. Further cleavage of the 40 kD N-terminal
fragment demonstrated that brain spectrin bound to the 20 kD proximal portion of
the rod domain in NF-L (Frappier et al., 1991). The binding of brain spectrin to
NF-L may indicate how NFs associate with the plasma membrane.
Plectin is an intermediate filament associated protein of an apparent
molecular weight of 300 kD (Wiche and Baker, 1982). Using in vitro binding
assays, plectin was shown to bind to the a-helical rod domain of all IF proteins
examined including the NF triplet protein subunits (Foisner et al., 1988). Plectin
has been extensively studied over the past decade in non-neural tissue (Wiche and
Baker, 1982; Wiche et al., 1983; Wiche et al., 1984) and is considered to be a
widely distributed IF associated protein. Plectin is believed to be important in the
interaction between IF proteins, and the association between IF proteins and other
components of the cytoskeleton such as the plasma membrane (spectrin), nuclear
envelop (lamins) and MTs via MAPs (Herrmann and Wiche, 1987). In addition to
these functions, plectin may be important in cell to cell interactions at tight
junctions or myotube attachment sites since both light and electron microscopic
show it associated with these regions (Wiche, 1989).
Synapsin I is a phosphoprotein located in the synaptic terminal and is
involved in neurotransmitter release (Llinas et al., 1985). Synapsin I is thought to
act as a cross-linker between synaptic vesicles and the cytoskeleton in synaptic
terminals thereby localizing vesicles near release sites in the terminal. It is thought
that upon influx of extracellular calcium, synapsin I is phosphorylated by CaM
kinase II and released from the cytoskeleton, thereby allowing synaptic vesicles to
associate with release sites on the presynaptic membrane (Hirokawa et al., 1989).
In vitro binding studies by Steiner et al. (1987) have shown that synapsin I binds
to NF-L. This interaction between synapsin I and NF-L can be reduced by 60%
upon phosphorylation of synapsin I with CaM kinase II. However, since NFs do
not appear to exist in polymer form at the synaptic terminal, the binding of NF-L
to synapsin I may not be functionally significant.
NAPA-73 is an avian specific 73 kD NF-associated protein that is expressed
developmentally in the nervous system and heart (Ciment et al., 1986; Ciment,
1990). In contrast to other NF binding proteins, NAPA-73 appears to associate
only with bundles of IFs (not individual subunits) suggesting that NAPA-73 is
involved in the bundling of filaments (Ciment, 1990).
NF binding to RNA and DNA was investigated by Traub et al. (1985). They
examined the binding of individual NF subunits to rRNA (total rRNA from Ehrlich
ascites tumor cells), native DNA (double stranded DNA), and heat-denatured DNA
(partially single stranded DNA) using sucrose gradients. NFs had a higher affinity
for 18S rRNA than 28S rRNA and this affinity could be abolished in the presence
of 6 M urea for NF-M and NF-H. In contrast NF-L interaction with 18S rRNA was
resistant to 6 M urea. Native DNA bound very weakly to NF subunits, whereas,
heat-denatured DNA bound strongly to NF subunits except in the case of NF-H.
The specificity of binding of the different forms of nucleic acids suggested that the
association was not solely based on electrostatic interactions (Traub et al., 1985).
The relevance of NFs binding to RNA and DNA is not known. However, some
researchers speculate that NFs degraded at the axonal terminal by calpain
proteases are transported back to the cell body where they could act as a
messenger for the events occurring at the nerve terminal (Traub, 1985; Schlaepfer,
Another characteristic aspect of NFs is the ability to bind calcium.
Assembled NFs in both phosphorylated and dephosphorylated states contain high
and low affinity binding sites for calcium (Lefebvre and Mushynski, 1987, 1988).
The dephosphorylation of NFs results in a 50% decrease in the low affinity calcium
binding sites which may be related to the loss of phosphate groups. Coinciding
with the decrease in low affinity calcium binding sites is an increase in the number
of high affinity binding sites. This increase in the high affinity calcium binding sites
is probably reflected by a change in the conformation of NFs after
dephosphorylation. Glutamic acid residues are known to constitute calcium binding
sites in such proteins as calmodulin (Kilhoffer et al., 1983), thus the glutamic acid
rich region in the C-terminus of NF tail regions may be the possible binding site for
calcium. Lefebvre and Mushynski (1988) found that the high affinity calcium
binding sites are located in the a-helical rod domain and that the low affinity
binding sites are located at the carboxyl-terminal region of NF molecule. The
functional significance of these calcium binding sites is unknown; however, two
possible functions for calcium binding to NFs have been suggested: (1) the ability
to regulate the activation of calcium binding proteases (calpain) which degrade NFs
in the synaptic terminal (Schlaepfer et al., 1985; Schlaepfer, 1988); and (2) the
buffering of calcium during nerve excitation (Abercrombie et al., 1990).
Relationship of NFs to Axonal Transport and Caliber
The transport of material from the cell body along the axon is an essential
neural function since the axon does not contain the cellular machinery for protein
synthesis. Anterograde axonal transport can be broken down into five components
on the basis of their uniform rate: (1) 250-400 mm/day (fast component); (2) 40
mm/day; (3) 15 mm/day; (4) 2-5 mm/day (slow component b); and (5) 0.2-1
mm/day (slow component a) (Willard, 1974). Cytoskeletal elements travel in slow
component a and b. Actin with its associated proteins travel in slow component
b, whereas, NFs and MTs, travel in the slow component a (Hoffman and Lasek,
1975). In addition, there appears to be a slower second wave of NFs travelling at
a non-uniform rate that is 100 fold slower than slow component a (Nixon and
Logvineko, 1986). It has been proposed that newly synthesized NFs travel down
the axon as assembled NFs, and are incorporated into the stationary cytoskeletal
network at random points during transport (Nixon and Logvineko, 1986). During
axonal transport, NF's overall charge become increasingly negative which
corresponds to an increase in phosphorylation state (Nixon et al., 1986). This
phosphorylation of NF-L and NF-M is short-lived; 50-60% and 35-40% of the
phosphates are removed in five days, respectively. In contrast, NF-H shows little
to no phosphate turnover during the same period (Nixon and Lewis, 1986). Lewis
and Nixon (1988) suggest that differential turnover rate of NF phosphorylation may
regulate the incorporation of NFs into the stationary NF network, and reflect a
dynamic interaction of NFs with other proteins.
Even though NFs are normally perceived as static structures, research
examining the role NF density as an intrinsic regulator of axonal caliber suggest
a more dynamic function for NFs (Hoffman et al., 1984). During development, the
increase in NF synthesis has been correlated to a decrease in NF transport
velocity and an increase in radial growth of the axon (Willard and Simon, 1983;
Hoffman et al., 1985a). This association of NF number to axonal caliber suggests
that regulation of NFs by gene expression and axonal transport largely determines
the size of the axon. Conversely, after a sciatic nerve crush, there is a decrease
in the number of NF proteins delivered to the axon which coincides temporally with
the reduction in axonal caliber proximal to the lesion in comparison to more distal
regions (Hoffman et al., 1985b). As the slow component advances at its normal
rate, the atrophy also advances (referred to as somatofugal atrophy), and when NF
synthesis returns to normal levels the atrophy is reversed (Hoffman et al.,1985b).
Similar results are observed with acrylamide toxicity (Gold et al., 1985), further
suggesting that NFs play an vital role in regulating axonal caliber.
In contrast, other research showed that NF density varies considerably
along the length of the axon, as well as in different axonal types, implying that a
local regulation of axonal cytoskeleton must occur (Berthold, 1982; Nixon and
Logvineko, 1986; Price et al., 1988; Szaro et al., 1990). To examine the role of
the cytoskeleton in the local modulation of axonal caliber, a dysmyelinating mutant
mouse line, Trembler, was used. The peripheral nerves of Trembler were
characterized by a reduction in axonal caliber, an increase in density of axonal
cytoskeletal elements, and a decrease in slow component axonal transport (Low
and McLeod, 1975; Low, 1976a,b; de Waegh and Brady, 1990). De Waegh and
co-workers (1992) examined the relationship of axonal caliber and NF
phosphorylation in the Trembler mouse and found that along with an increase in
NF density, there was a concomitant decrease in NF phosphorylation and axonal
caliber. Therefore, the decrease in NF phosphorylation could in turn be related to
a decrease in charge repulsion thus allowing the NFs to pack more closely
together. In contrast, De Waegh and co-workers (1992) suggested that these
alterations were regulated by the amount of myelin surrounding the axon. Grafts
of Trembler sciatic nerve placed into normal nerve showed regenerated axons with
the Trembler morphology while adjacent axons (control) had normal characteristics.
Recently, transgenic techniques were used to examine the effects of murine
NF gene overexpression on neurons and to determine if an increase in NF number
in the axon resulted in a concomitant increase in axonal caliber (Monteiro et al.,
1990). The transgene mRNA exceeded the endogenous NF-L mRNA by fourfold.
This resulted in a corresponding increase of the NF-L protein in the peripheral
axons of the trangenic mice. Although there was an increase in density of axonal
NFs seen morphologically, this did not result in a change in axonal caliber.
These results with overexpressed murine NF-L gene suggest that either the
other two NF subunits may be important in regulating axonal caliber or that the
correlation between axonal diameter and NF density may only be valid when there
is a decrease in NF synthesis. The latter possibility would lend support to the
theory of mechanical stability. Since NF-M and NF-H have unusually large
carboxyl terminal domains which protrude as sidearms from the core of the
filament and are highly phosphorylated in the axon, it has been proposed that NF-
M and NF-H are important in forming or regulating the spacing of NFs whether
through actual cross-bridges or charge repulsion from the highly negative carboxyl-
terminal domains. Although these data appear to contradict the active role of NF
density in controlling axonal caliber, it seems more likely that there are both
intrinsic and extrinsic factors involved and that the role of NFs seems to be more
prevalent in large myelinated axons (Hoffman et al., 1988).
Research on neuropathologies related to abnormal NF accumulation has
focused on deficits in axonal transport. Giant axonal neuropathies are
characterized as a focal accumulation of NFs along the axon (Gold et al., 1986)
which suggests the occurrence of a local interruption of NF transport resulting in
the accumulation of NFs. Two model systems have shown that with normal NF
synthesis, a retardation of slow component of axonal transport by B,B'-
iminodipropionitrile (IDPN) results in proximal axonal swelling and distal axonal
atrophy (Griffin et al., 1978; Clark et al., 1980); whereas, after treatment with 2,5-
hexanedione NF transport is accelerated, resulting in proximal axonal atrophy and
distal axonal swelling (Monaco et al., 1984 and 1989).
NF Expression After Axotomy
Differential changes in cytoskeletal proteins following axotomy of peripheral
nerves is observed for both sensory and motor neurons. Overall, the synthesis of
both tubulin and actin increases after axotomy whereas the synthesis of NF triplet
proteins are down regulated (Hoffman et al., 1987; Wong and Oblinger, 1987;
Goldstein et al., 1988; Tetzlaff et al., 1988; Oblinger et al., 1989). This results in
a decrease in the amount of newly synthesized NFs transported into the axon
(Oblinger and Lasek, 1988). However, based on immunocytochemistry, expression
of NF triplet proteins appears to remain unchanged The time course for changes
in synthesis is similar for all cytoskeletal proteins in that the largest effects occur
between 7 and 14 days, and in the case of a peripheral nerve crush, the level of
synthesis returns to normal between 3 to 8 weeks post injury. If reinnervation is
prevented, NF synthesis remains at decreased levels.
Normally, phosphorylated epitopes of the high molecular weight NF subunits
are restricted primarily to the axon whereas non-phosphorylated epitopes are
localized to the cell body and dendrites. However, after axotomy there is a
dramatic and transient increase in phosphorylated epitopes in the cell body that is
concurrent with a decrease in expression of NF mRNA (Moss and Lewkowicz,
1983; Dr&ger and Hofbauer, 1984; Goldstein et al., 1987; Shaw et al., 1988). As
labelling for NF phosphorylated dependent epitopes increase in the perikarya, there
is a concomitant decrease in labelling for the dephosphorylated dependent epitope
which suggests that phosphorylation is occurring at the sites that would normally
be recognized by dephosphorylated dependent NF antibodies (Goldstein et al.,
1987). In the case where axons do not normally regenerate, phosphorylated
epitopes on NF-H remain up to 60 days in the cell body after axotomy (Drger and
In contrast to the NF triplet proteins, peripherin expression is upregulated
after sciatic nerve crush in both dorsal root ganglion (DRG) cells and lumbar
motoneurons (Oblinger et al, 1989; Wong and Oblinger, 1990). In DRG cells,
peripherin is normally expressed in small diameter cells whereas NF triplet proteins
are localized primarily to the large.diameter cells (Parysek and Goldman, 1988;
Parysek et al., 1988: Ferri et al., 1990; Goldstein et al., 1991). After a peripheral
nerve crush, the mRNA level for peripherin increases in the large diameter cells
(maximizes at 7 to 14 days) but remains unchanged for the small diameter cells.
This increase in mRNA levels for peripherin translates into an increase in
immunoreactivity in large diameter DRG cells. As expected, NFs show a decrease
in mRNA synthesis which is not accompanied by a change in immunoreactivity in
the large diameter DRG cells. Similar results are observed in lumbar motoneurons
after sciatic nerve crush for peripherin. For peripherin, the mRNA levels increase
two fold after 4 days, remain elevated for 6 weeks, and finally recover to normal
levels at 8 weeks (Troy et al., 1990).
Role of NFs in Disease States
NFs are thought to be involved in the development of various
neuropathologies. The breakdown of the normal NF organization is prevalent in
motoneuron disease (Hirano, 1991), neurodegenerative disease (Goldman and
Yen, 1986) and toxin-induced neuropathologies (Griffin and Watson, 1988). In
amyotrophic lateral sclerosis (ALS) there is selective degeneration of anterior horn
motoneurons of the spinal cord. One of the hallmark features of ALS is an
abnormal accumulation of NFs in the perikarya, axons and dendrites as well as NF
induced swelling in the axons early in the degenerative process. It is yet to be
determined whether this increase in expression of NF protein is central to this and
other neuropathologies or is a secondary consequence of another cellular disorder.
Recent transgenic technology has resulted in two animal models to study
the overexpression of NFs, one that examines the effects of NF-L overexpression
and the other that examines NF-H overexpression. Doubly transgenic mice, which
resulted in a fourfold increase the murine NF-L gene, produced dramatic
phenotypic and morphological changes (Xu et al., 1993). Phenotypically, doubly
transgenic mice were one to two-thirds the body weight of age-matched controls,
kinetic activity was decreased and death usually occurred within 3 weeks due to
widespread skeletal muscle atrophy. In the CNS, murine NF-L expression was
restricted to neurons; however, outside the nervous system, the expression OF
murine NF-L was observed in different areas including skeletal muscle.
Morphometrically, select motoneurons showed chromatolytic features that included
a disrupted rough endoplasmic reticulum and a displaced nucleus. The
overexpression of murine NF-L resulted in excessive accumulation of NFs in the
soma and proximal axons of the anterior horn motoneurons which were undergoing
chromatolysis. In addition to the increase in NF density in large caliber axons, the
NFs were more closely packed together and were not always observed in the
normal parallel array to the long axis of the axon. A few mice lived past three
weeks, and after 2 months the axons showed a significant decrease in NF density
that were still higher than controls. Additionally, muscle mass returned and a slow
return of normal kinetic activity was observed. Near normal phenotype at nine
months occurred concomitant with a decrease in NF accumulation which had
plateaued at three to four weeks. These results led to the hypothesis that
excessive NF-L accumulation can result in motoneuron dysfunction before
widespread neuronal loss.
In a similar study, in which transgenic mice were produced with human NF-
H, a twofold increase of NF-H expression resulted in a more progressive
neuropathology that occurred over a period of three to four months (C6tM et al.,
1993). In these transgenic mice, the expression of the human NF-H was restricted
to neurons. Similar to ALS, progressive abnormal accumulations of NFs were
observed in the perikarya and proximal axons of anterior horn motoneurons of the
The excessive accumulations of NFs in the neuronal cell bodies in both
types of transgenic mice suggest that it is not one particular NF subunit is critical
to prevent normal cellular activity. However, the increase in density and
disorganization of NF structure taken together could interfere with normal axonal
transport and thus result in inappropriate accumulations of NF subunits throughout
the neuron. Thus, deficits in axonal transport appear to be a major component in
motoneuron neuropathies. This-may be due to indirect effects like an abnormal
increase in NF proteins in the soma that blocks normal axonal transport which may
be the case with ALS, or may be due to a direct effect of axonal transport, that
results in local accumulations of NFs, which may be the case with giant axonal
Overview of Dissertation
Although NFs are one of the most abundant proteins in the nervous system,
the dynamics of their function remain undefined. As in the case of other
cytoskeletal elements, such as MTs and MFs, one way of understanding or
defining function is realized by determining what types of proteins interact with
these cytoskeletal elements. In the set of experiments described in this
dissertation, I have undertaken this approach to identify candidate NF binding
proteins in an attempt to assign functional roles to NF proteins.
One candidate NF binding protein is a 300 kD protein which co-purifies with
IF proteins isolated from a crude spinal cord preparation. This protein was
identified as plectin, a protein previously characterized in non-neuronal tissue. The
relationship of plectin to its potential role in the nervous system, and specifically
to neural IF proteins was examined.
Using affinity column chromatography, additional candidate NF binding
proteins were identified based on molecular weight. A number of proteins bound
to the different NF affinity columns with varying specificity. One protein in
particular was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The in vitro binding of GAPDH to NF-L was examined as well as possible in vivo
interactions at the light and ultrastructural level.
These findings provide an initial framework for studying NF associated
proteins. NFs appear to interact with a number of proteins and these associations
may be specific to individual subunits as well as to the intact 10 nm filaments. In
addition, future studies that identify the other candidate NF associated proteins will
lead to a better understanding of NF function. Considering the differential labelling
of NFs in neurons, NF associated proteins may also show similar specificity which
may lead to important markers for different neuronal cell types along the neural
This chapter describes the routine biochemical and anatomical techniques
that were performed for this dissertation project. Techniques that were specific for
a particular chapter or modifications of these routine techniques will be described
in that chapter.
Lowry protein assay. The method of Lowry et al. (1951) was used for some
of the protein assays. Briefly, a standard curve from 0 to 100 pg bovine serum
albumin (BSA) to a final volume of 0.5 ml was made. The unknown concentration
of the protein solution was determined by diluting the protein solutions so it fell in
the range of the standard curve. Five milliliters of a solution containing 0.04%
copper sulfate, 0.08% potassium sodium tartrate and 2.94% sodium carbonate was
added to the BSA standards and protein samples to be assayed. The solutions
were then incubated at room temperature for 10 minutes. Next, 0.5 ml of 50%
Folin phenol reagent solution (diluted with deionized water) was added to each
tube and then each tube was incubated for 30 minutes at room temperature. The
absorbance was measured at 650 nm for each sample and the absorbance of BSA
standards was plotted against the known concentrations. The unknown protein
concentrations were determined from the linear regression line of the BSA
Pierce protein assay. Pierce Micro BCA protein assay kit was also used in
protein concentration determination because of its reliability and ease of use.
Protein concentrations were determined in the range of 0-20 pg/ml using BSA as
the standard and following the manufacturers instructions. Pierce Micro BCA
protein assay kit uses bicinchoninic acid which is a highly sensitive and selective
detection reagent for Cu'. The protein concentration of the unknown samples
were determined using the same methodology as described above.
SDS Polyacrylamide Gels
The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
technique was based on the method of Laemmli (1970) with an acrylamide to
bisacrylamide ratio of 37:1 (22.2%:0.6%). The electrophoretic apparatus used was
the Bio-Rad mini-protean II electrophoresis cell. Sample buffer containing SDS
and f3-mercaptoethanol (12.5% (v/v) 1 M Tris-HCI, pH 6.8, 4% (w/v) SDS, 10%
(v/v) glycerol, and 0.006% (v/v) bromophenol blue in ethanol) was added to each
sample and then each sample was boiled for 5 minutes before protein separation
on SDS polyacrylamide gels. The SDS polyacrylamide gels were run at a constant
current of 20 mA per gel. The gels were fixed in 40% methanol and 10% acetic
acid for 30 minutes, and then stained with 0.025% Serva Blue G for 1 hour
followed by destaining with 10% acetic acid for 1-2 hours. This method of staining
and destaining of gels is fast and efficient when compared to staining with
Coomassie Brilliant Blue R-250 (CBB) in methanol and acetic acid solution which
can take up to 24 hours to properly destain the background. In cyanogen bromide
(CNBr) cleavage experiments, CBB was used since the initial staining of the bands
is comparatively faster (15 to 20 minutes) and high background did not interfere
with accurately excising the gel band of interest.
Electroblotting of Proteins
Proteins were electrophoretically transferred to nitrocellulose or
polyvinylidine difluoride (PVDF) membrane using Bio-Rad Mini Trans-Blot Cell. A
submerged protein transfer system was used where the gel and membrane were
sandwiched between Whatman 3M paper and scrub pads. The transfer buffer
used in most situations contained 10 mM 2-[N-morpholino]ethanesufonic acid
(MES) pH 6.5, 0.01% SDS. The transfer lasted for 1-2 hours at 90 Volts
depending on the protein being transferred. To prevent overheating, the mini
trans-blot unit was immersed in an ice bath.
The proteins transferred onto the nitrocellulose membranes were visualized
with 0.025% Ponceau S in 40% methanol/10% acetic acid, and were used for
Western blots. Proteins transferred onto PVDF membranes were used for amino
acid analysis and were treated in a slightly different manner than nitrocellulose
membranes. Before proteins were transferred onto PVDF membrane, the
membrane was immersed in 100% methanol and then equilibrated in transfer
buffer for 20 minutes with two changes of buffer. After the transfer of proteins to
the PVDF membrane, the PVDF membrane was washed in deionized water for five
minutes, and stained in 0.1% CBB in 50% methanol for 5 minutes. The PVDF
membrane was partially destined in 50% methanol and 10% acetic acid for five
minutes, followed by a final wash in deionized water for 5 minutes (four changes
of water). The PVDF membrane containing the transferred proteins was dried and
stored at -200C until analysis.
Proteins were separated on SDS polyacrylamide gel and electrophoretically
transferred onto nitrocellulose membrane as discussed previously. The membrane
was blocked with 3% bovine serum albumin (BSA) or 5% nonfat Carnation instant
milk containing 0.1% Tween 20 in Tris buffered saline (TBS: 10 mM Tris-HCI (pH
7.5), 0.9% saline). If the nonfat milk was used to block the nitrocellulose, then the
primary and secondary antibody was diluted in this solution, and all but the last
wash was done with TBS containing 0.1% Tween 20 in place of TBS. The
remaining steps were the same as those described when BSA was used to block
the nitrocellulose membrane. The membrane was incubated with the primary
antibody containing 0.1% BSA for 1 hour at room temperature followed by three
10 minute washes in TBS. Next, the membrane was incubated with secondary
antibody, alkaline phosphatase conjugated anti-mouse or anti-rabbit, for 1 hour at
room temperature followed by two 10 minute washes in TBS. A ten minute wash
in developing buffer (10 ml 0.1 M Tris-HCI (pH 9.5) containing 5 mM MgCI2 and
0.1 M NaCI) followed. The antibody binding was visualized using 33pl 50 mg/ml
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 33pl 50 mg/ml Nitro Blue
Tetrazolium (NBT) in 10 ml developing buffer.
Protein Cleavage with Cyanogen Bromide
To cleave proteins at methionine residues, the cyanogen bromide (CNBr)
cleavage method was used on SDS polyacrylamide gel protein bands (Sokolov et
al., 1989). The protein to be cleaved by CNBr was separated on a SDS
polyacrylamide gel. The gel was stained with CBB (0.25% coomassie blue R-250
in 45% methanol and 10% acetic acid) and the gel band of interest was excised
from the gel. The gel band was dried down using a vacuum evaporator at 60-
800C. The volume to rehydrate the gel band was estimated by measuring the
length, height and width of the gel band before it was dehydrated, and that volume
was added to the dried protein gel band in the form of 200 mg/ml CNBr in 70%
formic acid. The gel band was incubated in the CNBr solution at 37C for at least
6 hours. The rehydrated gel band was dried down in the vacuum evaporator to
remove the remaining CNBr and its by-products. The dried cleaved gel band was
rehydrated and neutralized in 50% 2X sample buffer and 50% 1 M Tris for 15
minutes. The cleaved protein gel band was placed on 15% SDS polyacrylamide
gel to separate the protein fragments. The protein fragments were visualized using
Serva Blue G or silver stain depending on the protein concentration.
Isolation of Neurofilament Proteins
Crude Intermediate Filament Preparation
A crude intermediate filament (IF) preparation was used to obtain primarily
neurofilament (NF) proteins in vitro while maintaining some of their native
characteristics. Fresh spinal cord (specific species used will be discussed in the
relevant chapter) was homogenized gently using a Wheaton Dounce Type 40 glass
homogenizer for small preparations (510 g of tissue) or a Sears blender for larger
preparations (>10 g tissue). The tissue was brought up in 5 volumes of Buffer A
(0.1 M Tris-HCI (pH 7.5), 0.1 M NaCI, and 1 mM Na-p-tosyl-L-arginine methyl ester
(TAME)), and homogenized with 3 strokes of pestle 'B' followed by 1 to 2 strokes
with pestle 'A'. For larger preparations, the tissue was homogenized in blender at
low speed (three 5 second pulses) followed by three 5 second pulses at high
speed. The homogenate was made up to 10 volumes with Buffer A and
centrifuged at 2300xg for 10 minutes at 40C in a SS34 Sorvall rotor. The pellet
was taken up in 10 volumes 1 M sucrose in Buffer A, vortexed gently to break up
the pellet, and centrifuged at 2300xg for 20 minutes at 4C in a SM-24 Sorvall
rotor (small preparation) or a SS34 Sorvall rotor (large preparation). The floating
myelin layer, containing the NF bundles, was carefully removed and the volume
was made up to 5 volumes with Buffer A containing 1 M sucrose and 1% Triton
X-100. The myelin homogenate was gently vortexed and triturated to suspension,
and then shaken on an orbital shaker for 60 minutes at 40C. This allowed the
Triton X-100 to solubilize the myelin and release the intermediate filament bundles.
The suspension was centrifuged at 150,000xg for 1 hour at 4C in a Beckman TL-
100 rotor (small preparation) or a T865 Sorvall rotor (large preparation) to recover
the pelleted intermediate filament bundles. The pellet was composed mainly of NF
bundles and glial filaments. The pellet was taken up in the appropriate buffer and
stored at -200C.
Partially purified NFs were prepared using the method of Delacourte et al.
(1980). Fresh pig spinal cord (250 g) was homogenized in 400 ml of 0.1 M MES
(pH 6.5), 1 mM ethylenediamine tetraacetic acid (EDTA), 0.5 nM MgCl2, 1 mM
PMSF, and I mM TAME using a Sears blender at 4C. Initially, the spinal cord
was homogenized at low speed using 3 five second pulses followed by 3 five
second pulses at high speed. The crude homogenate was centrifuged at 13,500xg
for 45 minutes at 4C in a GSA Sorvall rotor. The supernatant was filtered through
8 ply gauze sponges type VII (Professional Medical Products, Inc.) to remove any
large floating debris. Next, the filtered supernatant was centrifuged at 78,000xg
for 30 minutes at 40C in a T865 Sorvall rotor. Glycerol was added to the
supernatant to form a final concentration of 20% (v/v) glycerol. The solution was
warmed to 37C for 20 minutes. The supernatant was centrifuged at 78,000xg for
45 min at 30C in a T865 Sorvall rotor. The pelleted material was combined and
homogenized in 50 ml of MES buffer and centrifuged at 147,000xg for 30 minutes
at 4C in a T865 Sorvall rotor. The pelleted material was a pale yellowish gel that
contained NFs and glial filaments. The pellet was stored at -200C.
Purification of Individual Neurofilament Subunits
Individual NF subunits were purified following a modified method of
Tokutake (1984). The pellet obtained from the NF Delacourte preparation was
homogenized on ice in 10 mM sodium phosphate monobasic (pH 7.0), 6 M urea,
1 mM EDTA, and 1 mM dithiothreitol (DTT) in order to solubilize the NFs into their
individual subunits. Insoluble material was removed by high speed centrifugation
(150,000xg) for 30 minutes at 40C using a T865 Sorvall rotor and the supernatant
was filtered through a 0.45 pm filter. The soluble material containing the NF
subunits was put through a DEAE cellulose column (DE-52) at a flow rate of 25
ml/hour at 4C. The DE-52 column was washed extensively to remove any
unbound proteins and before elution of bound proteins. To make sure no more
proteins were being eluted, the optical density at 280 nm of the wash buffer
coming through the column was checked. A linear gradient of 10 mM to 400 mM
monobasic sodium phosphate (500 ml) was used to elute the bound proteins.
Tokutake showed that varying the sodium phosphate concentration instead of
using a sodium chloride gradient resulted in a better separation of the individual-
NF subunits. Fractions of 5 ml were collected during the elution, and 10pl of each
even number fraction was separated on 7.5% SDS polyacrylamide gels to
determine which fractions contained the NF subunits. The fractions which were
purest (those that contained 98% of only one of the subunits) were pooled and
dialyzed against the appropriate buffer. The purified NF subunits were assayed
for protein concentration (Lowry or Pierce micro assay) and stored at -20C.
Anatomical Localization Experiments
Dorsal Root Ganglion Cell Cultures
New born rat pups (postnatal day 1) were killed by decapitation and the
body was pinned at the extremities to a piece of styrofoam. All instruments were
sterilized with 95% ethanol. The skin over the spinal cord was removed and a
longitudinal cut, using a scalpel, was made through the dorsal portion of the
vertebrae. With scissors the dorsal vertebral column was trimmed and the spinal
cord removed. The dorsal root ganglia (DRG) were located between the vertebrae
and resembled small white spheres. The DRG were removed one by one with fine
forceps from one side of the vertebral column and placed in a sterile microfuge
screw cap containing Liebowitz L-15 media (Sigma) with 10 ml/liter penicillin
(10,000 Units/ml), streptomycin (10,000 mag/ml) and amphotericin B (25 mag/ml)
(Gibco) (L-15 incomplete media). This was repeated for the contralateral side and
for the remaining rat pups so that each tube contained approximately 10 to 15
DRG. The ganglia were pelleted in a clinical centrifuge (setting 5) for 30 seconds.
To the pelleted ganglia, 240 pl 10X sterile trypsin-EDTA solution (Sigma; 0.5%
trypsin, 0.2% EDTA. 0.9% NaCl) was added. The resuspended DRG were then
incubated at 370C for 40 minutes to dissociate the ganglia. Then, the DRG were
centrifuged for 30 seconds at setting 5 in the clinical centrifuge, the trypsin
containing supernatant was removed, and 6-8 drops of fetal bovine serum (FBS;
Gibco) was added. The DRGs were incubated for 10 minutes at 370C, pelleted as
described above, and supernatant removed. DRG were triturated in 100 pl of L-15
incomplete media 15 times using an Eppendorf pipet to resuspend the ganglia into
single cells. Before plating, 500 pl of L-15 incomplete media was added to each
tube, then cells were plated on 6 acid washed coverslips (100 pl per plate) placed
in Petri dishes (35x10 mm). Two milliliters of L-15 complete media (L-15
incomplete media, 10% FBS, 0.6% glucose, 2 mM L-glutamine and 0.3% A4M
methylcellulose) was added to each Petri dish. Nerve growth factor (NGF) (Sigma
7S mouse submaxillary gland NGF) was added to result in a final concentration of
200ng/ml. The DRG cells were grown in a non-CO2 incubator at 370C for 24
Pheochromocytoma Cells (PC12 Cells)
A stock solution of PC12 cells was thawed rapidly at 37C, pelleted in a
clinical centrifuge, and resuspended in RPMI media containing 85% RPMI 1640
medium (Gibco); 10% heat inactivated horse serum, 5% FBS and 10 ml/liter of
penicillin (10,000 Units/ml), streptomycin (10,000 mag/ml) and amphotericin B (25
mag/ml) (Gibco). PC12 cells were plated on a collagen coated Petri dish (60x10
mm), grown at 37C in a water-saturated atmosphere containing 8.1% CO2, and
fed with new media every 2-3 days. When the cultures reached confluence, they
were subcultured at a ratio of 1:3 to 1:4. To generate neurite-bearing PC12 cells,
50 ng/ml 7S-NGF was added to each culture and the total serum concentration
was reduced to 1.5% from 15%. PC12 cells were feed every two days with RPMI
media containing 1.5% serum and 50 ng NGF. After about a week in culture,
PC12 cells produced neurites and were ready to be related on collagen coated
coverslips for immunocytochemistry. The PC12 cells were removed from the Petri
dish by trituration with a Pasteur pipette and pelleted in clinical centrifuge at low
speed where the supernatant was discarded (contains cellular debris including
detached neurites). The pelleted cells were resuspended in RPMI media (1.5%
serum) with 50 ng NGF, and plated onto coverslips. The PC12 cells produced
neurites within 8 to 16 hours and by 2 to 3 days the PC12 cells have formed
extensive neuritic processes.
Immunofluorescent Studies on Fresh Frozen Tissue
Adult rats, either Sprauge Dawley or Long Evans Hooded strains, were
anesthetized with 0.5 ml pentobarbital i.p. and sacrificed by decapitation. Nervous
system tissue was dissected out, frozen on dry ice for 1 hour or quickly frozen in
isopentane cooled with liquid nitrogen, and stored at -70C until sectioning. The
tissue of interest was cut at a thickness of 6 to 10 pm using a cryostat, placed on
petroleum ether cleaned glass slides, and stored at -20*C until they were labelled
with antibodies. Prior to incubation with primary antibodies, the tissue was fixed
in acetone (-20C) for ten minutes. Tissue sections were air dried 10 to 15
minutes and encircled with a PAP pen (The Binding Site) to make a well for the
antibodies. The two primary antibodies, one polyclonal and the other monoclonal,
were mixed together in buffer to obtain the appropriate final concentration for each
antibody and then were placed on the section. The tissue sections were then
incubated at 37C for 30 minutes or 4C for 24 hours, followed by three 10 minute
washes in PBS. Following primary antibody, sections were incubated for 30
minutes at 370C or 24 hours at 40C with secondary antibodies, FITC-conjugated
goat anti-mouse (1:40) and Texas Red conjugated donkey anti-rabbit (1:200).
After three 10 minute washes in PBS, the sections were coverslipped with anti-
bleaching agent (1.0 mg/ml para-phenylenediamine (Sigma) in 20 mM Tris-HCI pH
7.9, 90% glycerol). For tissue culture immunofluorescence, the DRG and PC12
cells were fixed in cold methanol (-200C) for 5 minutes. The remaining antibody
steps were as described above. The fluorescently labelled tissue sections were
visualized and photographed using a Zeiss Axiophot microscope system.
Immunohistochemistry on Formaldehyde Fixed Tissue
Adult rats, either Sprauge Dawley or Long Evans Hooded strains, were
anesthetized with either 0.5 ml pentobarbital or 80 mg/kg ketamine and 10 mg/kg
xylazine, and perfused with saline (0.9% NaCI) containing 0.05% (v/v) heparin and
0.05% (w/v) sodium nitroprusside followed by a fixative solution of 4%
paraformaldehyde in Sorensen's Buffer, pH 7.4 (19 mM monobasic sodium
phosphate, 81 mM dibasic sodium phosphate). The rat nervous system was
dissected out and placed in the above fixative solution overnight or PBS depending
on how well the tissue was fixed. Before sectioning, the tissue was blocked and
placed in phosphate buffered saline for at least 4 hours before being cut in 30 to
50 pm sections using a vibratome.
For immunocytochemistry, the tissue sections were incubated at in 0.3%
hydrogen peroxide in PBS for 30 minutes at room temperature to remove any
endogenous peroxidase activity. The tissue sections were washed three times for
10 minutes and mounted on chrome-alum coated slides and allowed to air dry for
at least one hour before beginning antibody staining procedure. Vectastain elite
ABC kit (KIT) was used to visualize the labelling of the primary antibody. The
tissue sections were blocked for 20 minutes in dilute normal serum (KIT) at 370C.
Excess blocking serum was shaken off and the primary antibody in 0.25% Triton-
X-100 was placed on the tissue section and incubated for 30 minutes at 37C or
24 hours at 4C. The tissue sections were washed three times for 10 minutes
followed by incubation with biotinylated secondary antibody (KIT) for 30 minutes
at 37C or 24 hours at 4C. The tissue sections were washed 3 times for ten
minutes followed by incubation with the avidin/biotin reagent (KIT) for 30 minutes
at 37C or 4 hours at room temperature. The tissue sections were washed 3 times
for ten minutes in PBS before being developed with freshly prepared
diaminobenzidine tetrahydrochloride (DAB) solution (0.05% DAB (Sigma),
containing 0.01% hydrogen peroxide in 0.1 M Tris, pH 7.2). The reaction was
monitored using an inverted microscope in order to determine when to stop the
reaction. The reaction was stopped by placing the tissue sections in tap water for
five minutes. The tissue sections were dehydrated using ascending alcohol
concentrations: 70%, 90%, 95%, 100%, 100%; and cleared in xylene and
coverslipped with permount.
THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE FILAMENT BINDING
PROTEIN, IN THE ADULT RAT CENTRAL NERVOUS SYSTEM
Although intermediate filament proteins (IFs) are found with various
polypeptides known as intermediate filament associated proteins (IFAPs), IFAPs
remain much less characterized than microtubule and microfilament associated
proteins. At present only a few proteins are known to be specific IF-binding
proteins, (Foisner and Wiche, 1991) such as plectin (Wiche, 1989), IFAP-300
(Yang et al., 1985; Lieska et al., 1985), NAPA-73 (Ciment et al., 1986), filensin
(Merdes et al., 1991), and filaggrins (Haydock and Dale, 1990).
One protein in particular, plectin, has been studied extensively in non-neural
tissue. Immunofluorescence studies of various non-neuronal tissues demonstrated
that plectin was localized to a number of different cell types, and has a cytoplasmic
distribution within the cell with a distinct tendency to be concentrated at the cellular
periphery near the plasma membrane (Wiche et al., 1983). Later work
demonstrated that plectin bound to immobilized IF proteins in vitro, including the
NF triplet proteins (Foisner et al., 1988). This interaction was found to involve the
a-helical rod domain in IFs and the a-helical rod domain of plectin (Foisner et al.,
1988; 1991). Originally, plectin was identified from rat glioma C6 cells as a major
component of Triton X-100 extracts with an apparent molecular weight of 300 kD
as determined from mobility on SDS-polyacrylamide gels (Pytela and Wiche, 1980).
Recent cloning and sequencing of rat plectin has shown that the actual
molecular weight of plectin is 527 kD (Wiche et al., 1991). Portions of plectin's
primary sequence are related to desmoplakin and the bullous-pemphigoid antigen,
both of which are found in association with IFs at the plasma membrane (Wiche
et al., 1991). Overall, plectin is postulated to function as a crosslinking protein,
mediating interactions between IF proteins and other components of the cell
(Foisner and Wiche, 1991) such as the plasma membrane via spectrin, the nuclear
envelope via nuclear lamins and microtubules via microtubule-associated proteins
(Koszka et al., 1985; Herrmann and Wiche, 1987).
As discussed earlier, previous research on plectin has focused on non-neural
tissue, and although plectin binds NF subunits in vitro, the in vivo significance of
this observation is not clear. This chapter focuses on demonstrating that plectin
is present in crude insoluble IF extracts from neural tissue and showing the
localization of plectin in the adult rat central nervous system.
A crude intermediate filament (IF) preparation from bovine and rat spinal cord
was obtained using a modified axonal floatation technique, details of this method
were described in Chapter 2 (Shelanski et al., 1971; Shaw and Hou, 1990).
Bovine spinal cord (50 g) was obtained from the slaughter house -1 hour
postmortem and prepared using the large preparation method. Rat spinal cord (-0.2
g) was obtained rapidly (>5 minutes) after decapitation and processed using the
small preparation method.
Partial purification of the 300 kD protein was accomplished using a Sephacryl
S-400 (Bio-Rad) column (1.5 cm x 100 cm). Bovine IF pellet was resuspended in
Buffer B (6 M Urea, 10 mM sodium phosphate (monobasic, pH 7.5), 1 mM EDTA,
1 mM TAME), and centrifuged at 150,000xg in a Sorvall T865 rotor for 45 minutes
at 4C to remove any insoluble material. The supernatant was filtered through a
0.8 pm filter followed by a 0.45 pm filter, then placed in a dialysis bag and
concentrated to a final volume of 2 ml by placing dry Sephadex G-50 around the
dialysis bag. The concentrated bovine IF material was separated on a Sephacryl
S-400 column which was equilibrated with Buffer B containing 5 mM DTT. The
flow rate was 15 ml per hour and 4 ml fractions were collected. Five pl from the
even number fractions were separated on 6% SDS polyacrylamide gels.
Proteins were separated on SDS polyacrylamide gel and transferred to
nitrocellulose using 10 mM MES (pH 6.8) and 0.01% SDS for 2 hours at 90 volts
(constant). For the crude IF preparation from rat spinal cord, approximately 3% of
final preparation was used per lane. A detailed description for the methods used
here were given in Chapter 2.
Monoclonal antibodies to plectin (1D8 and 1A2) and polyclonal serum to
plectin (p21) were previously characterized (Wiche and Baker, 1982; Foisner et al.,
1991). Monoclonal antibodies were from tissue culture supernatant and were used
either undiluted or diluted 1:2. The polyclonal serum, p21, was used at 1:50.
Alkaline phosphatase conjugated anti-mouse IgG secondary antibody was
purchased from Sigma and used at 1:1000 dilution. Biotinylated secondary
antibodies were obtained from Vector Laboratories and diluted according to the
manufacturers directions. Fluorescent secondary antibodies were obtained from
Jackson Laboratories and used at 1:100 dilution.
Both paraformaldehyde fixed and fresh frozen acetone fixed rat neural tissue
were used in this study. Plectin was localized in the paraformaldehyde fixed tissue
using the Vectastain Elite ABC kit. For fresh frozen acetone fixed tissue, plectin
was localized using the indirect immunofluorescence procedure. Both of these
anatomical methods were described in detail in Anatomical Localization section in
Besides the major IF proteins bands in a crude IF preparation from spinal
cord, there were a number of proteins that copurify with IFs (Figure 3-1a, b lanes
1). Since these proteins were present after salt and detergent extractions, it was
hypothesized that these proteins may functionally interact with IFs.
In particular, one high MW protein band at 300 kD on SDS polyacrylamide
gel, a relatively minor component in this IF preparation, was proposed to
correspond to plectin, a known 300 kD protein that interacts with IFs in vitro
(Foisner et al., 1988). The 300 kD protein from crude IF preparation of bovine
spinal cord was partially purified using Sephacryl S-400 column. Figure 3-1a
shows the crude bovine IF preparation (lane 1) and Sephacryl S-400 column
fractions 16-28 (lanes 2-8, respectively) in which lane 2 contains the 300 kD
protein. Immunoblot of fraction 16 (Figure 3-1a, lane 9) demonstrated that the
plectin monoclonal antibody ID8 labelled a 300 kD protein.
Since rat neural tissue was to be utilized in the immunocytochemical
localization of plectin in the CNS, I investigated whether plectin was present in rat
neural tissue and whether the plectin antibody cross-reacted with any of the other
proteins present in the crude IF preparation. Immunoblot of crude rat IF
preparation demonstrated that plectin was present in rat spinal cord, and the
antibody did not cross-react with any of the other proteins present in the rat
preparation (Figure 3-1 b). The weak smearing just below the major plectin band
which did not correspond to any protein band in the protein stained gel (Figure 3-
1b, lane 1) was probably a degradation product of plectin (Wiche, 1989). The
relatively low amount of plectin immunoreactivity detected may reflect a difficulty
in electrophoretically transferring plectin as a result of plectin's very high molecular
Figure 3-1. Identification of a 300 kD protein in bovine and rat crude IF spinal cord
(A) Partial purification of 300 kD protein (arrow) from crude IF preparation from
bovine spinal cord. Lane 1 is a 7.5% SDS polyacrylamide gel showing the IF
preparation before purification on a Sephacryl S-400 gel filtration column. Lanes
2-8 represent even number fractions (16-28) from the gel filtration column in which
fraction 16 contained the partially purified 300 kD protein. Lane 9 is an
immunoblot of fraction 16 labelled with monoclonal antibody to plectin (1D8). (H:
NF-H; M: NF-M; L: NF-L; G: GFAP).
(B) Immunoblot of rat spinal cord IF preparation showing plectin antibody staining.
Lane 1 is a 6% SDS polyacrylamide gel of a spinal cord cytoskeletal extract
stained with Serva Blue G. Lane 2 is an immunoblot strip labelled with plectin ID8
showing that the monoclonal antibody labels a 300 kD protein.
1 2 3 4 5 6 7 8 9
weight, at 527 kD. Even under optimal transfer conditions, when most of the other
high molecular weight proteins were transferred completely, a substantial portion
of the 300 kD protein band remained in the gel after electroblotting (data not
shown). In addition, it was possible that the fairly prominent 300 kD band seen in
SDS-PAGE contained other proteins (Lieska et al., 1985; Goldman and Yen, 1986)
besides plectin since ion exchange chromatography demonstrates that proteins
with a relative molecular weight of 300 kD are eluted off a DEAE ion exchange
column at different salt concentrations (data not shown).
Comparison of Plectin to IFAP-300
In addition to plectin, there was another IFAP identified which has similar
mobility on SDS polyacrylamide gel known as IFAP-300 (Lieska et al., 1985;
Goldman and Yen, 1986). It was of interest to compare the amino acid analysis
of these two proteins, since both plectin and IFAP-300 have similar mobilities on
SDS polyacrylamide gels (300 kD), staining distribution in non-neuronal cells, and
both bind to IF network. IFAP-300 amino acid composition data were taken from
Lieska et al. (1985) and the plectin amino acid composition was determined from
the published sequence (accession #S21876) and are presented in both table and
graphical form (Shaw, 1992) in Figure 3-2. Superficially, these two proteins appear
similar; however, differences were observed in the percent composition of a
number of amino acids Although these two proteins do not appear to be closely
related in amino acid composition, these results do not exclude the possibility that
there may be some sequence similarities, and since these two proteins have
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similar molecular weights, binding and tissue localization characteristics, plectin
and IFAP-300 may belong to a family of high molecular weight IFAP similar to that
observed with the high molecular weight microtubule associated proteins.
Overall, plectin immunoreactivity in the adult rat central nervous system was
predominantly associated with non-neuronal cells. In addition, plectin antibody
staining was present in a subset of neurons in the brainstem and spinal cord.
Immunoreactivity was generally strongest at the periphery of cells.
Telencephalon and diencephalon. Plectin immunoreactivity was absent in
neurons of the telencephalon and diencephalon. The only cells which labelled with
the plectin antibody in the cortical gray matter of the telencephalon were astrocytes
located in the ventromedial portion of the temporal cortex near the pial surface.
The density of astrocytic labelling with plectin antibodies decreased rapidly away
from the pial surface (Figure 3-3a). Although this labelling pattern may be
attributed to edge effect, a similar staining pattern was not observed in any other
cortical regions near the pia mater.
In the diencephalon, plectin positive astrocytes were observed at the
ventrolateral portion of the hypothalamus (Figure 3-3b). Plectin immunoreactivity
was found prominently in astrocytes throughout the white matter fiber tracts
including the optic tract (Figure 3-3c), corpus callosum, internal capsule, fornix, and
stria medullaris. Pronounced plectin immunoreactivity was observed in the
hypothalamus surrounding the third ventricle. Both ependymal cells lining the
Figure 3-3. Plectin immunoreactivity (ID8) of astrocytes in white and gray matter
of the telencephalon and diencephalon.
(A) The ventro-medial portion of the temporal cortex was the only cerebral cortical
area where plectin positive astrocytes were localized as visualized with DAB
(arrows). Scale bar: 25 pm.
(B) The ventro-lateral portion of the hypothalamus showed diffuse plectin
immunoreactivity of astrocytes(open arrow). Scale bar: 25 pm.
(C) White matter regions in the rat nervous system demonstrated plectin antibody
labelling of astrocytes as shown in the optic tract as visualized with
immunoflurescent secondary antibodies (open arrow). Scale bar: 25 pm.
dorsal portion and tanycytes, specialized ependymal cells, lining the ventral portion
of the third ventricle were plectin positive (Figure 3-4a). The plectin antibody
labelling of ependymal cells showed strong staining at the plasma membrane,
diffuse staining throughout the cytoplasm, and dense staining between the
ependymal cells (Figure 3-4b). The ependymal labelling pattern was observed in
all ventricles. A network of astrocytic fibers labelled with plectin antibodies was
seen in the subependymal layer (Figure 3-4b). In tanycytes, plectin
immunoreactivity was associated with the periphery of the cell body as well as with
the fibrous process (Figure 3-4c).
Cerebellum. Plectin immunoreactivity in the cerebellum was observed in
Bergmann glial fibers in the molecular layer, and in astrocytes in the granular cell
layer and white fiber tract layer (Figure 3-5a). The entire extent of Bergmann glial
processes were labelled with plectin antibodies; however, the strongest staining
was associated with Bergmann fibers terminal endings at the pial surface. The
relative intensity difference between the knob-like endings and processes of
Bergmann glia fibers was observed distinctly in a immunofluorescent higher power
view of molecular layer of cerebellum (Figure 3-5b). With respect to the terminal
endings of the Bergmann fibers which forms the external glial limiting membrane
in the cerebellum, it was difficult to determine whether these subpial structures
which were plectin positive formed conical and/or foot type endings (Palay and
Chan-Palay, 1974). The staining pattern once again showed strong labelling at the
periphery of the cells.
Figure 3-4. Plectin immunoreactivity (ID8) in the rostral hypothalamus surrounding
the third ventricle as visualized with DAB (3V).
(A) Cells lining the third ventricle are plectin positive and are of two types:
ependymal cells in the dorsal portion and tanycytes in the ventral portion. Scale
bar: 100 pm
(B) High power view of ependymal cells which demonstrates plectin antibody
staining throughout the cytoplasm with highest density of labelling at the cell
membrane (arrowheads). Light astrocytic staining is observed in the
subependymal cell layer just lateral to the ependymal cells. Scale bar: 25 pm.
(C) High power view of tanycytes which shows plectin immunoreactivity solely
around the plasma membrane (arrowheads) and radial process of the tanycyte
(arrows). Scale bar: 25 prm.
Figure 3-5. Plectin immunoreactivity (IA2 and ID8) in the cerebellum.
(A) In the cerebellum plectin antibody (IA2) labels the full extent of the Bergmann
glia process (arrow) in the molecular layer (ml) with pronounced labelling at the pia
surface (arrowhead) as visualized with DAB. Weak plectin immunoreactivity is
present in the granular cell layer (gcl) and appears to correspond to astocytic
processes. Note the absence of plectin antibody staining in the purkinje cell layer
(pcl). Scale bar: 100 pm.
(B) Higher power view of the cerebellum molecular layer showing the prominent
fluorescent labelling of the Bergmann glia terminal processes using ID8 antibody
(arrows) as visualized with immunoflurescent secondary antibodies. In comparison
the Bergmann glia fibers are weakly labelled with plectin antibody (open arrows).
Scale bar: 25 pm.
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Brainstem and spinal cord. The general pattern of the plectin antibody
staining pattern in the brainstem and spinal cord localized plectin primarily to non-
neuronal cells in the white matter tracts and to a subset of motoneurons. At low
magnification of the brainstem at the caudal medulla, plectin antibody labelling was
localized to astrocytes in the periphery and motoneurons in the nucleus ambiguus
(Figure 3-6a). A high magnification view of the ventrolateral aspect showed plectin
immunoreactivity in the inferior cerebellar peduncle and in the spinal tract of cranial
nerve V (Figure 3-6b). Plectin antibody labelling was pronounced at the outer
boundary of the inferior cerebellar peduncle as well as in glial fibers traversing the
inferior cerebellar peduncle. Throughout this region plectin positive astrocytes
were observed. Plectin staining was not observed in neurons associated with the
spinal nucleus of cranial nerve V (Figure 3-6b). In contrast, intense plectin
immunoreactivity was observed in motoneurons of nucleus ambiguus (Figure 3-6c).
However, there appeared to be a gradient in plectin antibody staining in
motoneurons such that some motoneurons labelled strongly while others stained
lightly (Figure 3-6c). This selective type of motoneuron staining was also observed
in the facial nucleus. In the pyramidal tract, plectin positive astrocytes were seen
as a network of fibers located adjacent to the pial surface (Figure 3-6d).
In the spinal cord, plectin immunoreactivity was observed at all levels with a
similar distribution. Plectin antibody labelling was observed in ependymal cells
lining the central canal, in astrocytes in the dorsal columns, in glial fibers
transversing the white matter, and in motoneurons in the ventral horn (Figure 3-
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7a). At a higher magnification, the ventral white matter showed plectin antibody
staining of glia cells which radially span from the pial surface to the gray matter
(Figure 3-7b). A closer examination of motoneurons in lamina IX of the spinal cord
gray matter showed that plectin immunoreactivity was associated with the cell body
and dendrites in a subpopulation of motoneurons (Figure 3-7c). Plectin staining
appeared to be excluded from the nucleus. In the vicinity of plectin positive
motoneurons, particulate staining was observed which may be cross-sections of
dendrites (Figure 3-7c.).
Choroid plexus and blood vessels. Choroidal epithelial cells showed
prominent plectin immunoreactivity (Figure 3-8a). Labelling with plectin antibodies
was observed throughout the cytoplasm with particularly intense staining at the
periphery and at points of contact between adjacent cells Differential plectin
antibody staining patterns were observed with blood vessels. Plectin
immunoreactivity was predominantly associated with endothelial cells of large blood
vessels (Figure 3-8b) but also was observed sporadically with smaller blood
vessels (Figure 3-8c).
These experiments were initiated in an attempt to identify a salt and detergent
resistant 300 kD protein band found in spinal cord cytoskeleton preparations which
could represent a NF binding protein. Biochemically, plectin was at least a
constituent of this 300 kD protein band, and was found in crude IF preparation of
Figure 3-7. Plectin immunoreactivity (ID8) in the cervical spinal cord.
(A) Low magnification view of the cervical spinal cord that demonstrates plectin
antibody labelling of astrocytes in the dorsal columns (dc), radially oriented glia in
white matter (wm) and motoneurons in the ventral horn (vh). Scale bar: 100 pm
(B) High magnification view of lateral white matter in which the intensity of plectin
antibody staining is graded. The strongest labelling is near the periphery (arrows).
Scale bar: 50 pm.
(C) High magnification view of motoneurons in lamina IX showing that plectin
immunoreactivity is associated with the cell body and processes of motoneurons
in the ventral horn. Particulate staining is observed in the vicinity of motoneurons
(arrowheads). Scale bar: 50 pm.
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Figure 3-8. Plectin immunoreactivity (p21) in choroid plexus and blood vessels.
(A) Choroidal epithelial cells show diffuse plectin antibody staining throughout the
cytoplasm and concentrated staining at the periphery of the cell (arrow). Scale
bar: 25 pm.
(B) Pericollosal artery labels strongly with the plectin antibody. Staining is localized
predominantly to the inner membrane of endothelial cells (open arrow). Scale bar:
(C) A smaller blood vessel in the temporal cortex labels positive with plectin
antibody. Note the astrocytic processes surrounding the blood vessel are labelled
with the plectin antibody. Scale bar: 25 pm.
the central nervous system.
One approach to begin to understand the role of a particular protein in the
nervous system is to examine the cellular distribution. Plectin immunoreactivity
was observed in a number of different cell types in the central nervous system with
varying staining intensities (Table 5-1). To summarize these data, prominent
plectin antibody staining was localized to the periphery of ependymal cells lining
the ventricles, to tanycytes, to choroidal epithelial cells, to endothelial cells and to
pia mater cells. In addition, dense plectin immunoreactivity was observed at
junctions between choroidal epithelial cells which may be related to tight junctions
forming part of the barrier between the blood and cerebral spinal fluid (CSF)
(Peters et al., 1991). Previous research by Wiche and co-workers (1989)
demonstrated that plectin was associated to various types of junctional zones. An
interesting and rather unexpected finding was that plectin antibodies labelled the
perikarya of a few neurons in the central nervous system. Generally, the staining
pattern of the plectin antibodies in non-neuronal cells of the nervous system
coincided well with previous work on the distribution of plectin outside the nervous
system (Wiche, 1989) and was similar to the vimentin staining pattern in the central
nervous system (Shaw et al., 1981; Pixley et al., 1981; Yen and Fields, 1981).
The previous studies of plectin concluded that plectin was widely but not
ubiquitously distributed with the peripheral regions of cells, with some overlap with
the IF pattern and submembraneous component. The cellular distribution of plectin
in the CNS appeared to be similar to that observed in non-neural tissue in that
Table 3-1. The distribution of plectin in different
cell types of the adult rat central nervous system.
Cell Type Staining
cortical pyramidal -
cortical interneurons -
cerebellar granule -
cerebellar Purkinje -
brain stem motoneurons +++/++/+/-
spinal cord motoneurons +++/++/+/-
choroid plexus +++
columnar epithelia +++
astrocytes (white matter) +++
astrocytes (gray matter) +/-
Bergmann fibers ++
endothelial linings +++/++/+/-
+: indicates that plectin was localized in these cells, and
number of + reflects intensity of staining; -: indicates that
plectin was not observed in these cells
plectin immunoreactivity was usually associated with the plasma membrane of the
A relationship of plectin with the blood-brain barrier (BBB) is suggested by a
tendency for plectin to be concentrated at all three of the elements of the BBB,
namely the pia, the walls of blood vessels and the linings of ventricles. However,
plectin immunoreactivity was not associated with all blood vessels and was only
infrequently associated with glial endfeet surrounding blood vessels. The BBB,
therefore, is not invariably associated with plectin immunoreactivity. Consequently
one can conclude that plectin is not absolutely required in all parts of this barrier.
In contrast, there is a consistent and strong layer of plectin immunoreactivity
associated with the ependymal layer including the choroid plexus, suggesting that
plectin may mechanically stabilize the submembraneous cytoskeleton and IF
components to form the three-dimension structure of the choroid plexus. In line
with this idea, previous ultrastructural studies have demonstrated a tendency for
plectin to be localized at interfaces between different tissues and especially
between tissues and fluid filled cavities. For example plectin is heavily
concentrated at the surfaces of kidney glomeruli, liver bile canaliculi, bladder
urothelium and gut villi (Wiche et al., 1983).
The selective staining pattern of plectin to subsets of motoneurons in the
brainstem and spinal cord is intriguing. One possible explanation for this unique
finding is that plectin may be associating with a particular NF subunit. Of the five
specific NF subunits, peripherin appears to be the best candidate. Peripherin has
a more selective distribution than the NF-triplet proteins and in contrast to cortical
localization of a-internexin, peripherin is localized to motoneurons of the spinal
cord and brainstem (Portier et al., 1984; Parysek and Goldman, 1988; Greene,
1989; Goldstein et al., 1991).
A common theme in this distinct plectin staining pattern is that plectin may be
associating with class 3 IF proteins (based on Steinert and Roop classification
scheme, 1988). Since peripherin and vimentin are similar in structure, it is likely
unlikely that plectin could be interacting at a similar site present in both proteins.
To shed some light on the selective cellular interactions of plectin in the nervous
system, Chapter 6 focuses on examining the distribution of plectin with IFs found
in the neural tissue.
Portions of this chapter are from a paper entitled "The Distribution of Plectin,
an Intermediate Filament Associated Protein, in the Adult Rat Central Nervous
System" by L.D. Errante, G. Wiche, G. Shaw. Journal of Neuroscience Research
37: 515-528. Copyrighted 1994 by Wiley-Liss. Text is used with the permission
of Wiley-Liss, a division of John Wiley & Sons, Inc.
CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM
Intermediate filaments (IFs) are a family of fibrous proteins found in most
eukaryotic cells and comprise a major component of the cytoskeletal network. In
addition to being organized into 6 classes based on sequence analysis (Steinert
and Roop, 1988), IFs can be categorized based on the types of cells they are
localized to. The strict cell-type specific expression of IFs, coupled with their high
abundance has permitted the extensive use of IF antibodies as convenient cell-
type specific markers. For example, in the nervous system, NF-triplet proteins, a-
internexin and peripherin are localized exclusively to neurons, and different neuron
types tend to have distinctly different IF composition (Shaw et al., 1981; Yen and
Fields, 1981; Portier et al., 1984; Pachter and Liem, 1985; Parysek and Goldman,
1988; Chiu et al., 1989). GFAP is localized to specific glia cell types (Yen and
Fields, 1981; Shaw et al., 1981) while vimentin can be found in subsets of glia,
ependyma and endothelia as well as some unusual neurons (Yen and Fields,
1981; Shaw et al., 1981; Draeger, 1983; Shaw et al., 1983; Schwob et al., 1986).
IF proteins are similar to the other major cytoskeletal proteins, microtubules
and actin, in that IFs have a number a polypeptides associated with them. Unlike
microtubule- and microfilament-associated protein, intermediate filament associated
proteins (IFAPs) are not well characterized especially in neural tissue. Due to the
lack of function associated with IFs in general, proteins have been classified as
specific IFAPs based on minimal criteria such that only one of the following
conditions need to be satisfied: (1) co-purification with IFs in vitro; (2) cellular co-
distribution with IFs; (3) binding to IFs or subunit proteins in vitro; and (4) have an
effect on filament organization or assembly (Foisner and Wiche, 1991).
Plectin is one of the few IFAP to have satisfied more than one of these
criteria including solid phase binding to all IF proteins examined and co-localization
with vimentin (Wiche, 1989). In the nervous system, plectin has been shown to
co-purify with crude IF preparations and to be localized in specific cell types that
contain IF proteins (Chapter 3). To address possible in vivo interactions between
plectin and IF proteins in the nervous system, we examined the co-distribution of
plectin with NF triplet proteins, peripherin, vimentin, and GFAP.
Experimental Tissue and Immunocytochemistry
For immunofluorescent studies, adult rats were sacrificed by decapitation.
Neural tissue was dissected out immediately, quickly frozen and stored at -70C.
Immunofluorescent procedures were described in Chapter 2.
For facial nerve axotomy, adult rats were anesthetized with methoxyflurane.
An incision was made just behind the right ear, the major branch of the facial nerve
was cut, and the incision was closed with surgical staples. Six to seven days post-
lesion rats were anesthetized with pentobarbital, and animals were killed by
decapitation or after intracardial perfusion with 4% paraformaldehyde as described
in detail in Chapter 2. In the case of the paraformaldehyde fix tissue, the
brainstem was blocked, immersed in 30% sucrose in PBS until it sunk and then
frozen on dry ice for 1 hour. Tissue was cut at 10 pm and placed in PBS and
treated in the same manner as previously described for paraformaldehyde tissue
Plectin monoclonal antibodies, clones 1 D8 and IA2 (diluted 1:2), and rabbit
polyclonal serum, p21 (diluted 1:50), were previously characterized (Wiche and
Baker, 1982; Foisner et al., 1991). NF-M rabbit polyclonal sera, R9 (diluted 1:200),
to the KE (lysine-glutamic acid) region of NF-M was made using NF fusion protein
RM:677-845. NF-H rabbit polyclonal antibody, R14 (diluted 1:200), to the KSP
(lysine-serine-proline) region of NF-H was made using NF fusion protein RH:559-
794 (Harris et al., 1991). Monoclonal antibodies against glial fibrillary acidic protein
(GFAP), clone GA5 (diluted 1:1000) and vimentin, clone V9 (diluted 1:200), were
purchased from Sigma (Debus et al., 1983; Osborn et al., 1984). Rabbit polyclonal
antibody to GFAP dilutedd 1:1000) was a gift from Dr. P. J. Reier. Peripherin
rabbit polyclonal sera, R19 and R20 (diluted 1:200) and mouse monoclonal
antibody, 8G2 (undiluted), were produced in our laboratory using a trp-E fusion
protein construct in the pATH expression vector which was a gift of Dr. Edward Ziff
(see Gorham et al., 1990). The fusion protein was expressed in Escherichia coli
in bulk and purified from inclusion body preparations by DEAE-cellulose ion
exchange chromatography as recently described for other trp-E fusion proteins
(Harris et al., 1991). The purified fusion protein was injected into rabbits and mice
to raise polyclonal and monoclonal antibodies using standard procedures (see
Initial characterization of peripherin antibodies were performed on crude IF
preparations from rat spinal cord to make sure that the antibody was not cross-
reacting with any other cytoskeletal proteins. Both peripherin monoclonal antibody
(8G2) and polyclonal antibody (R20) labelled, cleanly and specifically, a single
protein band at -57 kD. The peripherin protein band migrated at a slightly higher
apparent molecular weight than vimentin in crude IF spinal cord preparation (Figure
4-1, lanes 2-4, respectively). Peripherin polyclonal antibody R19 also showed the
same labelling of a single band at 57 kD (data not shown).
Co-localization in Cultured Cells
Dorsal Root Ganglion (DRG) Cells. Plectin antibodies labelled most of the
cell types in DRG cultures isolated from postnatal day 1 rat pups. The non-
neuronal cells or satellite cells consisted of two basic types; flat ameboid shaped
and spindle shaped which are illustrated in Figure 4-2. Plectin immunoreactivity
was localized throughout the cell body and the processes of the spindle shaped
Figure 4-1. Characterization of peripherin polyclonal and monoclonal antibodies.
Lane 1 shows a serva blue stained 6% SDS polyacrylamide gel of crude IF
preparation for rat spinal cord. Lanes 2 and 3 are immunoblots of this material
labelled with 8G2 and R20, respectively. These results show that both the
monoclonal and polyclonal antibodies to peripherin labelled a single protein band
at -57 kD. Lane 4 is an immunoblot of the same material labelled with vimentin
monoclonal antibody (V9) which demonstrates that the vimentin protein band is
located just below peripherin on a 6% SDS polyacrylamide gel.
57kD- -, -
G- 2 3 4
cells (Figure 4-2a, right side). In contrast the larger, flatter cells showed
particularly strong plectin immunoreactivity surrounding the nuclei and this labelling
intensity decreased dramatically at the peripheral regions of the cell (Figure 4-2a,
left side). Double labelling of these cells with vimentin revealed that plectin
immunoreactivity (Figure 4-2a) was localized discontinuously along vimentin
filamentous network (Figure 4-2b).
Plectin immunoreactivity in DRG neurons was similar to that observed for
the non-neuronal cells in tissue culture. Plectin antibodies strongly labelled the cell
body of DRG cells and weakly labelled the neuritic processes (Figure 4-3a);
whereas, peripherin labelled the cell body and neurites with similar intensity. The
peripherin antibody labelling pattern was consistent to that observed with NF triplet
protein antibodies in DRG (Shaw and Weber, 1981).
Phenocytoma (PC12) Cells. The distribution of plectin was compared to
NFs in both undifferentiated and differentiated PC12 cells. In undifferentiated
PC12 cells NF antibody labelling was found adjacent to the nucleus and in a tight
ball (Figure 4-3d), whereas plectin immunoreactivity was found diffusely throughout
the cell and with some overlap with the NF staining pattern (Figure 4-3c). After
exposure to nerve growth factor (NGF), PC12 cells stop dividing and differentiate
which results in the formation of neuritic-like processes as well as acquiring similar
properties as sympathetic neurons. In differentiated PC12 cells, plectin antibodies
labelled the cell body strongly with weak labelling in the processes that was similar
to that observed in the DRG cultures (Figure 4-3e). Additionally, plectin
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