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MICROSCOPY RESEARCH AND TECHNIQUE 41:416–430 (1998)
Localization of Focal Adhesion Kinase in Differentiating
Schwann Cell/Neuron Cultures
CRISTINA FERNANDEZ-VALLE,1, 2* PATRICK M. WOOD,3 AND MARY BARTLETT BUNGE3
1Department
of Molecular Biology and Microbiology and Center for Diagnostics and Drug Development,
University of Central Florida, Orlando, Florida 32816
2Orlando Regional Healthcare System/Health Research Institute, Orlando, Florida 32806
3The Chambers Family Laboratory of Electron Microscopy, The Miami Project to Cure Paralysis and the Departments of Neurological
Surgery and Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136
KEY WORDS
Schwann cell; basal lamina; myelination; focal adhesion kinase; immunogold
localization
ABSTRACT
Previous studies have shown that Schwann cells (SCs) differentiate into myelinforming or ensheathing cells only under conditions which allow the deposition of basal lamina and
extracellular collagen [Bunge (1993) Peripheral Neuropathy, pp. 299–316]. SC adhesion to basal
lamina is mediated by b1 integrins and function blocking antibodies to b1 integrins inhibit
myelination [Fernandez-Valle et al. (1993) Development 119:867–880]. Recently, focal adhesion
kinase (FAK), a cytoplasmic non-receptor tyrosine kinase, was found to mediate b1 integrindependent signalling in a variety of cultured cell types adhering to ECM components such as
fibronectin [reviewed in Schwartz et al. (1995) Ann. Rev. Cell Biol. 11:549–599; Ilic et al. (1997) J.
Cell Sci. 110:401–407]. In the present study, we have determined more precisely the respective time
courses of ECM deposition and myelination. In addition, we have studied by immunocytochemistry,
immuno-gold labelling, and electron microscopy the expression and subcellular localization of FAK
in nondifferentiating SCs and in SCs differentiating into myelinating cells. We show that the
development of basal lamina and extracellular collagen fibrils precedes by 3 days the appearance of
the first myelin sheaths. FAK was detected by immunocytochemistry or immuno-gold labelling only
in SCs differentiating in the presence of ascorbic acid. Localization of FAK to the abaxonal plasma
membrane was dependent upon ECM deposition. Cytochalasin D did not prevent or disrupt
localization of FAK to the plasma membrane. These data support the possibility that FAK acts as an
intermediate in the pathway by which basal lamina regulates SC differentiation. Microsc. Res.
Tech. 41:416–430, 1998. r 1998 Wiley-Liss, Inc.
INTRODUCTION
Extracellular matrix (ECM) is a rich, gelatinous
network of proteins and proteoglycans that cushions
cells, provides information about the environment,
forms a reservoir of trophic factors, and regulates cell
behavior by binding and activating cell surface receptors. In peripheral nerve, Schwann cells (SCs) and the
axons they ensheathe or myelinate are surrounded by
an ECM consisting of basal lamina and collagen fibrils.
During development, assembly and adhesion to basal
lamina regulates SC differentiation in response to
axons. The regulatory role of ECM is clearly seen
during differentiation of Schwann cells in vitro. SCs
cultured with sensory axons in a serum-free or serumrich (15%) medium contact axons but remain round and
undifferentiated (Moya et al., 1980; Fernandez-Valle et
al., 1993). Addition of ascorbate (50 µg/ml) to the
serum-containing medium induces SC differentiation
as evidenced by changes in cell shape and expression of
P0 mRNA that occurs within 2–5 days of ascorbate
supplementation (Eldridge et al., 1987; reviewed in
Bunge, 1993; Fernandez-Valle et al., 1993). Ascorbate is
essential for hydroxylation of proline and lysine residues in procollagen polypeptides. This post-translational modification allows procollagen molecules to
r 1998 WILEY-LISS, INC.
form triple helical structures that resist thermal and
proteolytic degradation when the mature collagen molecule is secreted into the extracellular space for selfassembly into collagen fibrils. Evidence that this is the
mechanism by which ascorbate promotes SC differentiation is provided by work using cis-hydroxyproline and
biochemical analysis of type IV collagen synthesized by
SCs grown in medium with and without ascorbate
(Eldridge et al., 1988, 1989). This work showed that
SC/axon interactions in the absence of ECM are not
sufficient to stimulate SC differentiation and suggests
that secondary regulatory or signaling mechanisms are
initiated by adhesion to ECM.
LAMININ AND b1 INTEGRINS PARTICIPATE
IN INITIATION OF SCHWANN CELL
DIFFERENTIATION
How does adhesion to basal lamina influence SC
differentiation? Basal lamina is composed of many
*Correspondence to: Department of Molecular Biology and Microbiology,
Biology Bldg. Rm. 306, University of Central Florida, Orlando, FL 32816–2360.
E-mail: cfernand@pegasus.cc.ucf.edu
Contract grant sopnsor: PHS; Contract grant numbers: 34499, 09923; Contract
grant sponsor: The Miami Project to Cure Paralysis; Contract grant sponsor:
State of Florida.
Received 4 December 1997; Accepted 12 December 1997
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
well-characterized molecules including collagen type
IV, laminin, entactin or nidogen, and heparan sulfate
proteoglycan. Various types of cell surface receptors
bind individual ECM components and mediate attachment to and signaling from the ECM. The identity of
the critical basal lamina component responsible for
signaling to the SC interior was investigated by adding
Matrigel (a commercial basal lamina preparation) or
purified basal lamina components to ascorbate-deficient medium in an effort to promote myelination.
Either Matrigel or purified laminin alone stimulated
myelination but collagen and heparan sulfate proteoglycan did not (Carey et al., 1986; Eldridge et al,. 1989;
Guenard et al., 1995). This suggests that laminin is the
component of basal lamina that induces or regulates
myelination.
The integrin family is the largest and most studied
group of ECM receptors. Integrins bind specific ECM
ligands and are capable of initiating signaling cascades
that regulate cell behavior (Clark and Brugge, 1995;
Juliano and Haskill, 1993; Kornberg and Juliano, 1992;
Richardson and Parsons, 1995; Schwartz et al., 1995).
Integrins are heterodimeric receptors consisting of
alpha and beta subunits. The possibility of many different combinations of subunits selected from 14 alpha
and 9 beta subunits provides a large repertoire of
ligand specificities for these receptors. For instance,
beta 1 integrin paired with alpha 5 binds fibronectin;
beta 1 paired with alpha 6 binds laminin; and beta 1
paired with alpha 1 binds both collagen and laminin.
The major integrin receptors expressed by undifferentiated and differentiating SCs are a6b1 integrin, a
laminin receptor, a1b1 integrin, a dual collagen/
laminin receptor, av, b3, and b8 integrins (Einheber et
al., 1993; Feltri et al., 1994; Fernandez-Valle, et al.,
1994; Milner et al., 1997; Niessen et al., 1994; Sonnenberg et al., 1990). When SCs are grown with neurons in
ascorbate-rich medium containing function-blocking b1
integrin polyclonal antibody, basal lamina is assembled
but is not bound to the SC surface and SCs fail to
myelinate axons (Fernandez-Valle et al., 1994). Sister
cultures grown in the presence of function-blocking
a1b1 monoclonal antibody myelinate normally. This
result is consistent with the hypothesis that laminin
binding to b1 integrin induces a signaling cascade that
‘‘primes’’ the SC to respond to instructive axonal signals
that specify the path of differentiation. Myelinating
SCs down-regulate a6b1 integrin and replace it with
another integrin laminin receptor, a6b4 integrin. a6b1
integrin has recently been shown to promote much of
the migration of mouse SCs on laminin (Milner et al.,
1997).
FOCAL ADHESION KINASE TRANSDUCES
SIGNALS FROM b1 INTEGRINS
IN OTHER CELL TYPES
Our knowledge of the immediate molecular consequences of b1 integrin activation is rapidly increasing,
and yet how these molecular events coordinate changes
in cell shape and gene expression during development
remain unclear. It is now recognized that b1 integrins,
upon binding their native ligands, initiate signal transduction cascades that affect cellular behavior and gene
expression in many cell types (Boudreau et al., 1995;
Juliano and Haskill, 1993; Kornberg et al., 1991; Streuli
417
et al., 1991; Werb et al., 1989; Yurochko et al., 1992). b1
integrins bind ECM ligands, aggregate, and induce
tyrosine phosphorylation of associated cytoplasmic proteins and recruitment of many signaling and structural
proteins to the plasma membrane at specialized sites of
ECM adhesion known as focal adhesions (Burridge et
al., 1988; Burridge and Chrzanowska-Wodnicka, 1996;
Jockusch et al., 1995). Focal adhesion kinase (FAK,
pp125) is a non-receptor tyrosine kinase identified as
the major protein tyrosine phosphorylated in response
to integrin activation in platelets and fibroblasts (Ilic et
al., 1997; Lipfert et al., 1992; Schaller et al., 1992).
There is indirect evidence that FAK is involved in the
transduction of signals from integrin receptors, which,
unlike many growth factor receptors, do not contain
inherent kinase domains. This evidence derives from in
vitro binding studies using peptides that mimic b1
cytoplasmic domains and extracts of cells over-expressing FAK (Schaller et al., 1995). FAK-b1 integrin interactions, however, have not been demonstrated in whole
cells adhering to ECM in vitro or in cells undergoing
differentiation. Recent studies have identified numerous proteins that bind to FAK and participate in
down-stream signaling events (Schlaepfer et al., 1994;
Miyamoto et al., 1995). These include the src kinase
family (e.g., as fyn kinase) and regulatory or docking
proteins such as Cas and paxillin (Hanks and Polte,
1997). Recruitment of additional proteins such as Grb2
to the focal adhesion complex allows interaction with
the well known ras/mitogen-activated kinase pathway
(Schlaepfer et al., 1994; Miyamoto et al., 1996; Schlaepfer and Hunter, 1996). Therefore, FAK is likely to play
important roles in regulating growth and differentiation of many cell types (Hanks and Polte, 1997; Ilic et
al., 1997).
ROLE FOR F-ACTIN IN b1
INTEGRIN-DEPENDENT SIGNALING
MECHANISMS
The actin cytoskeleton provides the immature cell
with motility and the mature cell with structural
integrity. Actin is a dynamic structure that, through
interactions with other proteins, forms an organizational network determining the localization of signal
transduction molecules (Carraway and Carraway, 1995;
Mochly-Rosen, 1995) that bind to cell surface receptors
involved in regulating cellular growth and differentiation. Recruitment of F-actin to the juxtamembrane
focal adhesion complex occurs during b1 integrinmediated signal transduction via this complex. b1
integrins interact with the actin cytoskeleton and this
interaction appears to be an integral part of signaling
(Lipfert et al., 1992; Otey et al., 1990; Schaller et al.,
1992; Shaw et al., 1990). In all cell types studied,
disruption of F-actin by cytochalasin D (CD) inhibits
tyrosine phosphorylation of FAK, an early and key step
in b1 integrin-dependent signal transduction that triggers recruitment of additional signaling molecules to
the focal adhesion complex (Lipfert et al., 1992; Miyamoto et al., 1995; Richardson and Parsons, 1995;
Schaller et al., 1992; Schaller, 1996). F-actin, however,
is not required for FAK-b1 integrin co-localization
(Miyamoto et al., 1995).
When a low CD concentration is added to SC/neuron
cultures, SCs assemble and adhere to basal lamina,
418
C. FERNANDEZ-VALLE ET AL.
elongate, and engulf axons, but do not myelinate or
express myelin specific mRNAs encoding cyclic nucleotide phosphodiesterase, MAG, and P0 (Fernandez-Valle
et al., 1997). This finding suggests that the initial
changes in cell morphology indicative of SC differentiation can be uncoupled from changes in gene expression.
A possible mechanism for this effect is the disruption,
by CD, of FAK activation as explained above. As an
initial test of this possibility we have studied the
expression and co-localization of FAK in SCs cocultured with neurons in undifferentiating and differentiating conditions and in the presence of low doses of
CD. A striking increase in FAK imunostaining was
observed when SC/neuron cultures were treated with
ascorbate to induce differentiation. Ultrastructural immunogold staining was employed to determine the
localization of FAK in differentiating SC/neuron cocultures. Gold labelling was observed in the cytosol and
juxtamembrane at sites of basal lamina adhesion in
differentiating and myelinating SCs and in CD-treated
SCs that form basal lamina but do not myelinate (this
work has appeared in abstract form, Fernandez-Valle,
1996.)
MATERIALS AND METHODS
Antibodies
Two FAK antibodies, a mouse monoclonal (catalog no.
05–182, Upstate Biotechnology, Inc., UBI, Lake Placid,
NY) and a rabbit polyclonal (catalog no. sc557, Santa
Cruz Biotechnology, Inc., SCBI, Santa Cruz, CA) were
used for immunocytochemistry and immuno-gold labelling.
Tissue Culture
Primary SC Cultures. The Brockes et al. (1979)
method was used to isolate SCs from sciatic nerves
removed from E21 or newborn Sprague-Dawley rats
(Charles River, Raleigh, NC). SCs were expanded in
vitro in medium containing Dulbecco’s modified Eagle’s
medium (DMEM), 10% heat inactivated fetal bovine
serum (FBS; Gibco/BRL; Grand Island, NY), forskolin
(Sigma; St. Louis, MO), and pituitary extract (BTI;
Stoughton, MA) on poly-L-lysine (Sigma) coated 100
mm tissue culture dishes. SC cultures were passaged
no more than four times before the SCs were plated
onto sensory neuron cultures.
Sensory Neuron Cultures. Neurons were isolated
from cervical dorsal root ganglia of Sprague-Dawley rat
embryos at 15 days of gestation by dissociation with
trypsin. Cells were plated on poly-L-lysine and laminincoated (Gibco/BRL) glass coverslips (Carolina, Burlington, NC) and maintained in Eagles’ minimum essential
medium (EMEM) containing human placental serum (a
generous gift from Dr. R. Devon, Saskatoon, Canada) or
FBS, nerve growth factor (NGF) and glucose as described in Fernandez-Valle et al. (l993). SCs and fibroblasts were eliminated using one pulse of the antimitotic agent, fluorodeoxyuridine (10 µm). Additional
details of the culture procedure are provided in Kleitman et al. (1991). Neuron-only cultures were maintained 7–14 days in serum-containing medium (EMEM,
10% FBS) before immunostaining for FAK.
SC/Neuron Cultures. SCs were removed from culture dishes using trypsin, washed extensively in L-15
containing 10% serum to inactivate trypsin, and rinsed
in L-15 (Gibco/BRL). Approximately 30,000 SCs were
seeded onto purified neuron cultures and maintained
for 1 week in serum-only medium to allow the cultures
to become fully populated with SCs (Fernandez-Valle et
al., 1994). Cultures were switched from serum-only
medium to serum-plus-ascorbate medium (EMEM, 15%
FBS, NGF, 50 µg/ml ascorbate) for 7–10 days to allow
myelination.
Cytochalasin D Treatment. SC/neuron cultures
were grown in serum-only medium for 1 week to expand
the SC population on axons. All cultures at this time
have equivalent SC densities. Cultures were then fed
serum-plus-ascorbate medium with or without CD
(Sigma, St. Louis, MO) at 0.25 µg/ml. Medium was
replenished every other day until myelin became visible in the control cultures (approximately 7–8 days
later) and then cultures were processed as described
below.
Immunocytochemistry
Cultures were processed for immunochemistry as
previously described (Fernandez-Valle et al., 1997).
Briefly, all cultures were rinsed in PBS, fixed in PBS/4%
paraformaldehyde (PFA) for 10 minutes, and permeabilized with 0.2% Triton X-100 in PBS/4% PFA. SC/
neuron cultures grown in serum-plus-ascorbate medium were additionally permeabilized with a 50%/100%/
50% series of acetone at 220°C to increase antibody
penetration through ECM. After blocking non-specific
reactions by incubating in PBS/10% normal goat serum, FAK monoclonal antibody (UBI) and/or P0 antibody (a generous gift from Dr. J. Brockes) at 1:100
dilution was added for 1 hour, then rinsed, and goat
anti-mouse or rabbit secondary antibody conjugated to
rhodamine or fluorescein (Organon Teknika Corp., West
Chester, PA) was added for 30 minutes. Control staining was carried out by omitting the primary antibody.
Cultures were rinsed, post-fixed, and mounted in an
anti-fade solution (Citifluor, Canterbury, England) containing Hoechst nuclear dye 33342 (Sigma). Cultures
were viewed on a Zeiss (Thornwood, NY) universal
microscope.
Electron Microscopy
Standard Fixation and Embedding. Cultures
were fixed in buffered glutaraldehyde followed by osmium tetroxide, dehydrated in ethanol, and embedded
in Embed (Electron Microscopy Services, Fort Washington, PA; Ratner et al., 1986). Areas for examination
were selected, thin sectioned, and stained with uranyl
acetate and lead citrate. Sections were viewed with a
Philips (Mahwah, NJ) CM10 electron microscope.
Quantitation of Developmental Time Course.
SC/neuron cultures were switched from serum-only to
serum-plus-ascorbate medium to initiate differentiation into myelin-forming cells. At daily intervals thereafter, cultures were fixed and processed for electron
microscopy and the percentage of SCs with myelin,
basal lamina, and fibrillar collagen was determined. SC
differentiation was assessed by analysis of 10 electron
micrographs/time point. A minimum of 10 of the largest
axons/micrograph were selected and the presence of
myelin, basal lamina, and collagen fibrils associated
with the SC contacting each selected axon was recorded.
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
Immunogold Fixation and Embedding. SC/neuron cultures were fixed and embedded in LRGold
(Electron Microscopy Sciences, Fort Washington, PA)
using the method reported by Gillespie et al. (1994).
Briefly, cultures were immersed in 4% PFA in 0.1 M
phosphate buffer (pH 7.4) containing 0.01 M sodium
periodate, 0.075 M lysine, and 3% sucrose for 2 hours at
room temperature. Fixed cultures were washed several
times in 0.1 M phosphate buffer with 3% sucrose (rinse
buffer) and stained in 0.25% tannic acid in the same
buffer for 30 minutes at 4° C. Subsequent steps were
carried out at 4° C. Cultures were rinsed several times
in rinse buffer and then incubated for 30 minutes in
rinse buffer containing 50 mM ammonium chloride to
quench aldehydes. Cultures were washed 3 times in 0.1
M maleate buffer (pH 6.2) containing 4% sucrose and
then stained with 2% uranyl acetate in maleate/sucrose
buffer for 30 minutes. Cultures were dehydrated in a
graded ethanol series from 50 to 90% at 220°C. Cultures were infiltrated for 30 minutes in a sequential
series of 1:1 ratio of LRgold:ethanol, 7:3 ratio LRgold:
ethanol and, finally, LRgold with 0.05% benzoin methyl
ether for 30 minutes before embedding by inverting the
coverslip cultures onto a gelatin capsule filled with
resin and polymerizing at -20°C for 24 hours with UV
light (365 nm). Glass coverslips were removed by
immersion in hydrofluoric acid. Areas of the embedded
cultures were thin sectioned and collected onto nickel
grids.
Immunogold Staining. Grids were incubated for
30 minutes to 2 hours in a blocking solution containing
10 mM Tris pH 7.4 buffer, 500 mM sodium chloride, 1%
bovine serum albumin (BSA), 0.5% fish skin gelatin,
0.05% Triton X-100, and 0.05% Tween 20 at room
temperature. Primary antibodies were diluted in blocking buffer (UBI monoclonal, 1:140; SCBI polyclonal,
1:500) and grids were inverted over a 20 µl drop of
anti-FAK antibody solution and incubated overnight at
4°C in a humidified chamber. The following day grids
were washed several times in blocking buffer and
incubated in a 1:25 dilution of 10 nm gold-conjugated
goat anti-mouse or anti-rabbit secondary antibody (Ted
Pella, Inc., Redding, CA) for 1 hour. Grids were washed
extensively with distilled water, fixed with 2.5% glutaraldehyde in PBS for 10 minutes, washed in a stream of
distilled water and then exposed to osmium vapors for
15 minutes. Grids were stained in lead citrate for 2
minutes and viewed with a Philips CM10 electron
microscope.
Quantitation of Gold Particles. Two to five areas
from SC/neuron cultures grown in serum-only or in
serum-plus-ascorbate medium with and without CD for
8 days were selected to assess immunogold labelling
with FAK antibodies. Areas were randomly selected
and photographed, all gold particles present in 5–25
electron micrographs of each section were counted, and
the distribution was recorded. The distribution of all
gold particles in multiples of 2 or more are reported.
Evaluation of Nonspecific Labelling. With the
monoclonal UBI antibody, 14–35% of single gold particles fell on the plastic resin and not on cellular
structures. In 5 of 7 labelling experiments, clusters of
two or more gold particles were not observed on the
resin and, in the other two experiments, only 8 and 9%
419
of aggregates of 2 or more gold particles were observed
on the plastic resin. SCBI polyclonal antibody used at
1:500 dilution resulted in low background staining and
specifically labelled the same structures observed with
the monoclonal antibody in PBS. Single gold particles
were most often found dispersed on the plastic sections
and in cultures grown in serum-only medium. Their
distribution was not included in this report to reduce
the possibility of including non-specifically labelled
sites. Changing the blocking buffer from TBS plus BSA
and detergents to PBS with 0.1% acetylated BSA, 0.1%
Tween 20, and 0.05% Triton X-100 to reduce nonspecific antibody binding abolished all labelling. Additional experiments are required to carefully titrate
detergent and acetylated BSA concentrations to completely eliminate nonspecific antibody binding. Both
antibodies labelled SC nuclei and occasionally the
nuclear envelope. Because nuclear labelling was observed in undifferentiated SC/neuron cultures, it also
was considered non-specific binding of the primary
antibodies. All labelling was abolished when primary
antibody was omitted, indicating that background labelling observed was not due to the gold-conjugated secondary antibody. Structures grouped under ‘‘other’’ in
Tables 1–3 included unrecognizable structures, cell
surface, and collagen fibrils.
RESULTS
Extracellular Matrix Deposition
Precedes Myelination
ECM deposition, visible as basal lamina and collagen
fibrils, precedes SC differentiation. This was clearly
seen in the results of a 7-day ultrastructural time
course study (Fig. 1). The results demonstrated that
basal lamina and collagen fibril deposition occurred
simultaneously and preceded the onset of myelination
by 3 days. At the time of ascorbate addition (day 1), SCs
had been growing in continuous contact with axons in
serum-containing but ascorbate-lacking medium for 7
to 10 days; fewer than 20% of the SCs exhibited ECM
and SCs had not yet myelinated axons. Ultrastructural
analysis of these cultures revealed that SCs contacted
axons but extended few processes into axon fascicles
(Fig. 2). Previous work in our laboratories has shown
that even after 3–5 weeks of co-culture with sensory
neurons in serum-only medium, SCs do not differentiate. Instead, they continue to proliferate in response to
axons and only a few myelin segments (0–10 myelin
segments/culture) form.
By 4 days after ascorbate addition to the medium,
basal lamina and collagen were observed in periodic
patches along the SC surface in nearly 40% of the
SC/axon units (Fig. 1). Axon fascicles had begun to be
subdivided by SC processes and substantial amounts of
basal lamina and collagen fibrils had formed, increasing the space between axons and neighboring SCs.
Typically, a number of SCs were engulfing axons and
isolating them from other axons (Fig. 3). A rare thin
myelin segment was observed. During the subsequent
24 hours, there was a dramatic increase in ECM
deposition, so that at day 5, 80% of the axon/SC units
displayed basal lamina and collagen fibrils. During this
rapid increase in ECM deposition, several additional
thin myelin segments appeared (Fig. 4a). Many more
420
C. FERNANDEZ-VALLE ET AL.
Fig. 1. Deposition of basal lamina and
collagen fibrils is concurrent and precedes
myelination in Schwann cell/neuron cultures. Quantitative development time course
showing the appearance of basal lamina,
collagen fibrils, and myelin sheaths following
addition of serum-plus-ascorbate to SC/N cultures.
SCs were differentiating and were engulfing, spiraling,
or in one-to-one relationships with axons. There was
increased space between SCs occupied by basal lamina
and collagen fibrils and large axon fascicles were less
frequently observed. The number of axon/SC units with
myelin increased slowly with time (Figs. 1 and 4b),
reaching 20% of the total by 7 days, a time by which
90% of axon/SC units exhibited ECM. The myelin
sheaths present were now of greater width than those
observed at 4 days, and basal lamina covered the
myelinating SC exterior, which was associated with
increased fibrillar collagen. The developmental sequence of basal lamina and collagen deposition followed
by initiation of myelination suggests that interaction of
the SC with ECM signals or regulates further differentiation. To explore this possibility, we have studied the
pattern of expression and localization of FAK during
the period preceding and the period coinciding with
myelination (Fernandez-Valle, 1996).
FAK Is Detected by Immunostaining in
Differentiating and Myelinating SCs
Cultures of sensory neurons grown alone and SC/
neuron cultures grown in serum-only or serum-plusascorbate medium were immunostained with FAK antibodies to determine if SCs express FAK and whether
FAK expression is dependent upon ECM deposition.
Immunocytochemical analysis failed to detect FAK in
the soma or neurites of dorsal root ganglion neurons
cultured alone (Fig. 5a,b) or in SCs cultured with
neurons in serum-only medium for 2 days (Fig. 5c,d).
FAK was readily detected in the perinuclear cytoplasm
and bipolar cytoplasmic extensions of differentiating
SCs grown 5–8 days in serum-plus-ascorbate medium
(Fig. 5e,f). FAK continued to be expressed at high levels
in myelinating SCs and was present along the extent of
the myelin internode (Fig. 5g,h). FAK was also observed
in the perinuclear cytoplasm of non-myelinating SCs in
cultures grown 21 days in serum-plus-ascorbate medium (Fig. 5i,j). Double immunostaining confirmed that
FAK and P0, the major protein in peripheral myelin,
were co-expressed in myelinating Ss (Fig. 6).
The striking increase in FAK immunostaining that
occurs following ascorbate addition suggests that FAK
expression might be induced or up-regulated by ascorbate. However, in a companion study, Western blot
analysis was used to verify FAK expression. Using this
technique and a different FAK antibody suitable for
immunoblot analysis of denatured protein, FAK was
found to be expressed in SCs cultured alone, and in
undifferentiated SC/neuron cultures growing in serumonly medium, as well as in SC/neuron cultures grown 4,
7, 14, and 21 days in serum-plus-ascorbate medium
(Chen and Fernandez-Valle, unpublished data). FAK
was not detected in sensory neuron cultures, confirming the immunostaining results. The Western blot
results indicated, therefore, that FAK was expressed at
all stages of SC development in vitro and its expression
was not dependent upon ECM deposition or axonal
contact.
FAK Is Present at Juxtamembrane Sites of Basal
Lamina Adhesion in Differentiating and
Myelinating Schwann Cells
A comprehensive evaluation of FAK-gold labelling
was carried out to determine the subcellular localization of FAK. FAK localization was studied in cultures
grown in serum-only medium and in serum plus ascorbate medium. In serum-only cultures (Fig. 7a), multiple
gold particles only infrequently labelled the abaxonal
juxtamembrane regions or SC processes, in contrast to
differentiating and CD treated cultures (Table 1). Gold
labelling was never observed in sections from differentiating 8 day SC/neuron cultures reacted with only the
gold-conjugated secondary antibody (Fig. 7b).
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
421
Fig. 2. Schwann cells grown with sensory neurons in serum-containing but ascorbate-lacking medium
do not differentiate. Fascicles of axons with few intervening SC processes are observed. Only scant basal
lamina and collagen fibrils are present, allowing close apposition of neighboring SCs (arrows). X31,000.
In differentiating cultures, the consistently labelled
subcellular structures were the cytosol and abaxonal
juxtamembrane regions of SCs and SC processes of
differentiating SCs that were associated with basal
lamina and collagen fibrils (Fig. 8a,b). At times, the gold
particles labelled sub-plasmalemmal densities as well.
Because these structures were only rarely labelled in
undifferentiated SC/neuron cultures grown in serumonly medium, this FAK labelling was considered to be
specific. The labelled plasma membrane domain was
usually adherent to a nascent basal lamina patch. In
one oblique section through a myelinating Schwann
cell, a large gold aggregate was observed in the cytoplasm adjacent to plasma membrane that was apposed
to collagen fibrils and, by inference, basal lamina (Fig.
8a, inset). In myelinating SCs, multiple gold particles
were observed in the outer cytoplasmic collar, just
exterior to the outermost myelin lamella (Fig. 8c). The
distribution of all gold particles in multiples of two or
more in cultures 8 days after adding differentiation
medium is shown in Table 2. In Table 2, the organelle
labelled by the SCBI polyclonal antibody was a clathrin-
422
C. FERNANDEZ-VALLE ET AL.
Fig. 3. Schwann cells grown with sensory neurons for 4 days in serum plus ascorbate medium begin
differentiation. Many large and small diameter axons are ensheathed by SCs. There are two examples (*)
of a SC ensheathing only one axon, a stage prefatory to myelination. Basal lamina and collagen fibrils
have appeared in relation to the SC surface (arrows). X31,000.
coated vesicle associated with a 14 gold particle cluster.
Occasional labelling of clathrin-coated vesicles and
granular endoplasmic reticulum was observed using
the UBI monoclonal antibody. The significance of this
labelling is not known and more extensive experiments
will be necessary to determine whether labelling of
these organelles is specific. Occasional axon and myelin
labelling was also observed.
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
423
Fig. 4. Schwann cells begin to form thin myelin sheaths 3 to 4 days
after ascorbate addition. a: Five days after ascorbate addition, a few
Schwann cells elaborated myelin around the larger diameter axons,
and other Schwann cells are in a 1:1 relation with axons or are
ensheathing multiple small diameter axons. Increasing amounts of
basal lamina and collagen fibrils are present. b: Eight days after
ascorbate addition, many well-myelinated axons are present. Basal
lamina and collagen fibrils completely surround the axon/Schwann
cell unit. With time, progressively more Schwann cells will complete
differentiation into myelinating or ensheathing cells. X23,000.
FAK Is Present at Juxtamembrane Sites of Basal
Lamina Adhesion in Schwann Cells Grown With
Sensory Neurons in the Presence of
Cytochalasin D
Immunogold staining to detect FAK was also carried
out in cultures grown in serum-plus-ascorbate medium
containing CD that disrupts actin polymerization and
myelination but does not disrupt basal lamina deposition. SC/neuron cultures grown in low CD concentrations fail to develop myelin, but assemble basal lamina
and begin differentiation as assessed by changes in SC
morphology, i.e., elongation and formation of the first
Fig. 5.
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
425
Fig. 6. FAK and P0 are co-expressed in myelinating Schwann cells. Phase (A) and fluorescent double
immunostaining for FAK (B) and P0 r in SC/neuron cultures grown 21 days in serum plus ascorbate
medium. The majority of P0-positive SCs express FAK that is localized along the entire length of the
myelin internode. X400.
SC membrane spiral around axons (Fernandez-Valle et
al., 1997). In these cultures gold particle labelling of
elongated SCs was frequently observed. Triplets of gold
particles labelled the abaxonal juxtamembranous regions of SCs engulfing axons or in the process of
elongation (Fig. 9). The labelled plasma membrane
domains adhered to basal lamina patches, which were
in turn associated with collagen fibrils. Subplasmalemmal densities in SCs were also frequently labelled by
multiple gold particles (Fig. 9, inset). The distribution
of gold particles is shown in Table 3. As in SC/neuron
cultures grown in the absence of CD, the predominant
structures labelled in CD-treated SCs were the cytosol,
cellular processes and abaxonal juxtamembranous regions of elongating or spiraling SCs.
DISCUSSION
The results reported here provide new clues about
the role of ECM in regulating SC differentiation. These
results include: (1) the demonstration that basal lamina
and collagen fibrils form at the same time, 3 days
preceding myelination; (2) the demonstration that SCs
treated with ascorbate exhibit strongly increased immunostaining for FAK; (3) the demonstration that differentiating SCs exhibit cytosolic and juxtamembrane localization of FAK; and (4) the demonstration that
myelinating SCs display FAK localization in the outer
cytoplasmic collar of the myelin sheath.
Fig. 5. FAK is expressed in differentiating and myelinating
Schwann cells. Phase (a,c,e,g,i) and fluorescent (b,d,f,h,j,) images of
FAK immunostaining in neuron-only cultures (a,b) and in SC/neuron
cultures grown 2 days in serum-only medium (c,d) or in serum-plusascorbate medium for 5 days (e,f) and 21 days (g–j). Neurons grown
alone and SCs grown with neurons for 2 days in serum-only medium
do not express FAK. SCs express FAK after 5–7 days of growth in
serum-plus-ascorbate medium (single SC at arrow in f). In myelinating SCs, FAK staining is present along the myelin internode (arrow in
h) and, in non-myelinating SCs, FAK is observed in the perinuclear
area (arrow in j). a–d, X1,000; e–j, X600.
Whereas there is compelling evidence that basal
lamina deposition regulates SC myelination, a quantitative time course study to determine the chronological
relationship between the appearance of basal lamina
and myelin sheaths had not been done. Additionally,
the temporal development of collagen fibrils in the
culture system had not been studied previously. The
results of this study demonstrated that basal lamina
and collagen fibrils form at the same time, and that
their appearance begins within 1 day of ascorbate
addition to the culture medium. Initially, ECM appears
as intermittent patches along the SC surface and with
time more SCs relate to ECM and the ECM becomes
more complete. There is a 3-day delay between initiation of ECM deposition and the appearance of the first
myelin segments. ECM assembly clearly precedes myelination; this sequence of developmental stages is
consistent with the hypothesis that ECM assembly
functions to regulate steps of SC differentiation that
occur later.
FAK Expression in Schwann Cells
Immunostaining results suggested that FAK was not
expressed by sensory neurons or by SCs cultured with
sensory neurons in serum-only medium that does not
support myelination. Immunoblot analysis of culture
extracts confirmed that sensory neurons do not express
FAK, but indicated that FAK is present in SC cultures
and in SC/neuron cultures grown in serum-only medium (Chen and Fernandez-Valle, unpublished data).
Our interpretation of these results is that undifferentiated SCs express FAK when in contact with neurons,
but that the antigenic epitope recognized by the antibodies used for immunostaining is masked in some way.
FAK was detected by Western blot analysis using a
different antibody recognizing denatured FAK. The
time course for the appearance of FAK immunostaining
in SC/neuron cultures following addition of ascorbate to
serum-only medium parallels the onset of SC differentiation and suggests that the FAK epitopes become
426
C. FERNANDEZ-VALLE ET AL.
Fig. 7. a: FAK labelling is not observed in Schwann cells grown
with sensory neurons in serum-only medium. Single gold particles are
dispersed within SC cytoplasm and nucleus but specific multiples of
gold are not observed adjacent to plasma membrane (b). Photomicro-
graph of an elongating SC grown in serum-plus-ascorbate medium for
8 days and incubated with only the secondary gold conjugated
antibody. No gold labelling is observed in the absence of the FAK
antibody. X45,000.
unmasked as SCs deposit basal lamina. This ‘‘unmasking’’ could result from a change in FAK localization or
association with other cytoplasmic proteins. Results of
the Western blot experiments did not indicate an
increase in FAK expression associated with switching
SC/neuron cultures from serum-only to serum-plus
ascorbate medium.
The distribution of FAK immunogold labelling near
the plasma membrane at sites of basal lamina adhesion
in differentiating SCs is consistent with the expected
427
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
TABLE 1. Distribution of gold particles ($2) in Schwann cell/neuron cultures grown 8 days in serum-containing
but ascorbate-lacking medium
Exp.
no.
1
2
Ab
Total gold/#
photos
Axon
Cytosol
SC
process
JxPlasma
membr.
Organelles
Nucleus
Other
UBI
UBI
26/11
41/8
2 (7%)
4 (10%)
2 (7%)
5 (12%)
0
8 (20%)
0
2 (5%)
0
3 (7%)
18 (69%)
6 (15%)
4 (15%)
13 (32%)
Fig. 8. FAK is present in juxtamembranous sites of basal lamina
adhesion in differentiating and myelinating Schwann cells. a: A large
cluster of gold particles is located just within the cell at the site of
basal lamina adhesion in a myelinating SC grown 8 days in serum-plusascorbate medium. This area, enclosed in the rectangle, is enlarged in
the inset. Collagen fibrils, located just beyond the basal lamina, are
shown in oblique section (see inset). b: In a cross-sectional view of a
SC cytoplasmic process engulfing an axon, 4 gold particles are present
just inside the abaxonal plasma membrane associated with ECM.
Axons are occasionally labelled with single gold particles. c: Gold
particles are seen in the cytoplasmic collar just outside the myelin. a,b,
X55,000; c, X100,000.
location of FAK in other cell types. Cultured cells form
focal adhesions at sites of cell-ECM attachment through
integrins (Burridge et al., 1988). At this site, structural
and signalling proteins accumulate beneath the plasma
membrane and initiate signalling cascades that regulate cell growth and differentiation (Jockusch et al.,
1995). FAK is a cytoplasmic non-receptor tyrosine
kinase that is recruited to the membrane and is rapidly
phosphorylated on tyrosine residues as a result of
integrin binding of ECM components such as fibronectin and laminin (Burridge et al., 1992; Schaller et al,
1992; Schaller, 1996). Previous work in this field indicates that FAK and b1 integrins co-localize at the
plasma membrane and autophosphorylation of FAK
428
C. FERNANDEZ-VALLE ET AL.
TABLE 2. Distribution of gold particles ($2) in Schwann cell/neuron cultures grown 8 days in serum- and ascorbate-containing medium
Exp.
no.
1
2
3
4
Ab
Total gold/#
photos
Axon
Cytosol
SC
process
JxPlasma
membr.
Organelles
Nucleus
Other
UBI
UBI
UBI
SCBI
289/26
81/23
61/5
87/7
24 (8%)
0
4 (7%)
0
49 (17%)
23 (28%)
23 (38%)
25 (29%)
16 (6%)
9 (11%)
4 (7%)
8 (9%)
59 (20%)
7 (9%)
6 (10%)
10 (11%)
22 (8%)
4 (5%)
4 (7%)
14 (16%)
56 (19%)
24 (30%)
16 (26%)
18 (21%)
37 (13%)
14 (17%)
4 (7%)
5 (6%)
Fig. 9. FAK is present in juxtamembranous sites of basal lamina
adhesion in Schwann cells grown with sensory neurons in the presence of cytochalasin D. Frequently, three linearly arranged gold
particles may be seen just within the plasma membrane of elongated
SCs or SC processes engulfing or spiraling around axons. Multiple gold
particles (2–7) often labeled subplasmalemmal densities at sites of
basal lamina (inset). X100,000; inset, X110,000.
leads to recruitment of additional signalling molecules
to the focal adhesion complex (Burridge and Chrzanowska-Wodnicka, 1996; Miyamoto et al., 1995). CD
inhibits FAK phosphorylation and down-stream signalling events but not the co-localization of b1 integrin and
FAK (Lipfert et al., 1992; Miyamoto et al., 1995). The
distribution of gold labelling is consistent with the
known function of FAK. Gold was observed in the
cytosol, consistent with a cytoplasmic kinase, and at
the membrane at sites of adhesion to ECM. Labelling of
sub-plasmalemmal domains adhering to basal lamina
in CD-treated cultures is consistent with the observation that FAK-b1 integrin co-localization is maintained
in cells treated with CD. The occasional labelling of
organelles may represent additional sites of FAK recruitment as has been shown for other kinases such as src
which is found in endosomes (Mochly-Rosen, 1995).
Our findings support a role for FAK in b1 integrinmediated signalling involved in regulating SC differentiation into myelin-forming cells. FAK expression in
myelinating SCs that do not express b1 integrin was
surprising because, thus far, FAK has only been reported to interact with b1 integrin. FAK localization in
myelinating SCs was observed at the plasma membrane, and sometimes in the outer cytoplasmic collar
adjacent to the myelin lamellae. These results suggest
429
FAK LOCALIZATION IN DIFFERENTIATING SCHWANN CELLS
TABLE 3. Distribution of gold particles ($2) in Schwann cell/neuron cultures grown 8 days in serum-, ascorbate-,
and cytochalasin D-containing medium
Exp.
no.
1
2
Ab
Total gold/#
photos
Axon
Cytosol
SC
process
JxPlasma
membr.
Organelles
Nucleus
Other
UBI
UBI
86/18
13/7
14 (16%)
0
20 (23%)
0
7 (8%)
3 (23%)
20 (23%)
2 (15%)
0
0
19 (19%)
5 (38%)
6 (6%)
3 (23%)
that FAK has a role independent of b1 integrinmediated signalling and could be involved in maintaining the myelinating phenotype. Additional work will be
necessary to determine the function of FAK in myelinating SCs.
ACKNOWLEDGMENTS
We thank April Bradford, a MARC summer fellow,
Antje Feucht, Jason Tanner, and Marcella Schlein for
assistance with experiments; Margaret L. Bates, J.-P.
Brunshwig, Ernesto Cuervo, and Anna M. Gomez for
technical help; Robert Camarena for photographic reproduction; and Charlaine Rowlette for word processing.
We thank Dr. J. Brockes for his generous gift of Po
antibody. This work was supported by PHS grants
34499 to C.F.V. and 09923 to R.P.B., The Miami Project
to Cure Paralysis, and the State of Florida.
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