Department of Pathology & Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
* Author for correspondence (e-mail: yurchenc{at}umdnj.edu )
Accepted 28 November 2001
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Summary |
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Key words: Schwann cell, Laminin, Collagen, Integrin, Dystroglycan, Utrophin
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Introduction |
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A model of the assembly of basement membranes on cognate cell surface
membranes is that laminin and type IV collagen receptors provide surface
anchorage, concentrating them and facilitating their self-assembly into ECM
heteropolymers (Colognato et al.,
1999; Colognato and Yurchenco,
2000
). A number of investigators have proposed that both
ß1-integrin and dystroglycan play critical roles in the mediation of
assembly in various tissues (Brakebusch et
al., 2000
; DiPersio et al.,
1997
; Henry and Campbell,
1998
; Lohikangas et al.,
2001
). However, it is unclear if these particular receptors
constitute the cell surface anchors that mediate assembly
(Cote et al., 1999
;
Sasaki et al., 1998
).
-Dystroglycan is a mucinous glycoprotein that is noncovalently bound to
transmembrane ß-dystroglycan and present on Schwann cells. It is thought
to link the LG modules of
1- and
2-laminins to the cytoskeleton
through its interaction with cytoskeletal dystrophin and/or utrophin (reviewed
by Henry and Campbell, 1996
).
Schwann cell integrins capable of interacting with laminins are
1ß1,
6ß1 and
6ß4, the first binding
through
1-domain VI and the latter two binding through G-domain
(Colognato et al., 1997
;
Colognato and Yurchenco, 2000
;
Einheber et al., 1993
;
Yurchenco et al., 1997
). The
Schwann cell expression pattern of these receptors changes during development
and in response to specific physiological processes
(Fernandez-Valle et al., 1994
;
Jaakkola et al., 1993
;
Stewart et al., 1997
).
Although their signaling mechanisms have been intensively studied, integrin
roles in basement membrane formation are not well understood.
In this study we have investigated the contributions of dystroglycan,
ß1-integrin, and corresponding interacting long arm globular LG modules
of laminin to Schwann cell basement membrane formation in vitro. Untreated
Schwann cells, regardless of age, were found to express little, if any,
endogenous 1- or
2-laminins on their surfaces. Later passaged
Schwann cells became competent for basement membrane assembly, a process
initiated by exogenous laminin. We found that dystroglycan and utrophin
underwent a dynamic rearrangement with exogenous laminin into a dense
(reticular) structure with classical ultrastructural features of a basement
membrane. Formation of this architecture depended upon the participation of
the terminal two laminin LG modules and polymerization. Moreover, collagen
type IV was absent from the structures and was able to co-assemble into
basement membrane-type structures in laminin-dependent manner when added
exogenously. By contrast, ß1-integrin did not appear to be required for
basement membrane assembly and instead mediated the formation of a separately
defined fibrillar extracellular matrix (ECM). Finally, comparison of competent
with incompetent cells implicates a third receptor or surface-anchoring
molecule required for basement membrane assembly on Schwann cell surfaces.
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Materials and Methods |
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Proteins and antibodies
EHS laminin-1, placental laminin-2/4 and collagen IV were purified as
described (Cheng et al., 1997;
Yurchenco and O'Rear, 1994
).
Laminin proteolytic fragments E3, E4, E8 and E1' were prepared as
described (Yurchenco and O'Rear,
1994
). Non-polymerizing laminin was prepared by treatment with
AEBSF as described (Colognato et al.,
1999
). Rabbit polyclonal anti-laminin-1 antibody, anti-laminin-2/4
antibody, anti-E4 and anti-E8 antibodies against specific laminin proteolytic
fragments were prepared by immunizing rabbits with the respective fragments.
Each serum was affinity-purified on columns of immobilized fragment followed
by cross-absorption against the other fragments as described
(Yurchenco et al., 1993
) and
used at 10 µg/ml. Rat monoclonal anti-
1 chain of laminin antibody
were used at 1:125 dilution (Upstate Biotechnology Inc, Lake Placid, NY).
Hamster ß1-integrin-specific IgM antibody Ha2/5 (BD Pharmingen, San
Diego, CA) were used at a final concentration of 10 µg/ml. Mouse monoclonal
-dystroglycan-specific antibody IIH6 were used at a 1:2 dilution of HB
101 hybridoma media (a kind gift of Kevin Campbell, University of Iowa).
Monoclonal anti-Utrophin antibody (MANCHO3) were kindly provided by G. E.
Morris (N. E. Wales Institute, UK) and were used at a 1:3 dilution for
immunostaining. Monoclonal S100-specific antibody (DAKO Corp, Carpinteria, CA)
were used at 1:100. Polyclonal goat anti-collagen type IV antibody were used
at 1:100 (Southern Biotechnology, Birmingham, AL). FITC- and Cy3- conjugated
secondary antibodies were from Sigma (St Louis, MO) and Jackson
Immunochemicals (West Grove, PA) and were used as recommended.
Immunocytochemistry
Cells grown on 16-well slides, following various treatments, were rinsed
three times with 10 mM sodium phosphate, pH 7.4, 127 mM NaCl (PBS, phosphate
buffered saline), 0.5% bovine serum albumin (BSA) and fixed in 3%
paraformaldehyde (EM Science, Gibbstown, NJ) in PBS for 10 minutes at room
temperature. Cells were permeabilized 0.5% Triton X-100 in PBS, 0.5% BSA for
15 minutes on ice when staining of intracellular epitopes was desired. For
detection of surface-bound or exposed proteins the detergent step was omitted
with exeption of Ha2/5 antibody. Blocking with 5% goat serum was performed for
30 minutes at room temperature. Cultures were then stained with various
primary and appropriate secondary antibodies conjugated with either FITC or
Cy3. All antibodies incubations were performed in PBS, 0.5% BSA, 0.5% goat
serum for 1 hour at room temperature. Control staining was performed using
standard primary IgG or IgM instead of specific ones. Slides were mounted in
DAFCO mounting medium (Sigma-Aldrich) and imaged using an Olympus IX-70
inverted microscope fitted for epifluorescence and phase-contrast. Digital
images were captured with a Princeton Instruments 5-mHz Micromax cooled-CCD
camera fitted into the left side port.
Immunocytochemistry quantitation
Surface area covered by laminin or type IV collagen was calculated using
IPlab 3.0 software. Seven random low magnification (20x) microscopic
fields for each condition were photographed at the same exposure settings. A
segmentation range was chosen based on immunofluorescence intensity and
maintained for all conditions evaluated in the data set. Quantification of
area coverage in the now highlighted segments was performed and normalized for
the total cell area for each field.
Electron microscopy
Cells were plated onto 8-well Permanox chamber slides (Nalgene Nunc) 1 day
before the experiment. Inhibiting agents (IIH6 Ab, Ha2/5 Ab and laminin
fragments) were added to cell cultures 20 minutes before the addition of 10
µg/ml of laminin. Laminin, AEBSF-laminin and no laminin controls were
performed in parallel. Cells adherent to plastic were fixed in 0.5%
gluteraldehyde and 0.2% tannic acid in PBS for 1 hour at room temperature, and
then transferred to modified Karnovsky's fixative (4% formaldehyde and 2.5%
gluteraldehyde containing 8 mM CaCl2 in 0.1 M sodium cacodylate
buffer, pH 7.4). Samples were washed with PBS and post-fixed in 1% osmium
tetroxide in 0.1 M sodium cacodylate buffer, pH 7.4 for 1 hour to produce
osmium black. Samples were then dehydrated through a graded series of ethanol
and embedded in Epon/SPURR resin (EM Science) that was polymerized at 65°C
overnight. Both thick (1 µm) and thin sections (90 nm) were cut with a
diamond knife on a Reichert-Jung Ultracut-E ultra-microtome. Thick sections
were stained with 1% methylene blue in 1% sodium borate for light microscopy
and thin sections were stained with saturated uranyl acetate (20 minutes)
followed by 0.2% lead citrate (2.5 minutes). Images were photographed with a
Jeol JEM-1200EX electron microscope.
To address the effect of blocking reagents on basement membrane, parameters were established to quantify the extent of electron-dense structures that formed on cell surfaces exposed to medium. Three different depth levels, separated by up to 1 mm, were cut into the cell block for each sample to generate thin sections, with each thin section containing 5-10 cells. Fifteen electron micrographs were prepared for each level, taken randomly. The 45 micrographs from the same sample were randomly divided into three groups to permit determination of data scatter. Linear electron-dense (lamina densa) structures with a length of 2 µm and over were considered continuous. Short stretches of lamina densa-type matrix that were less than 2 µm in length were considered as discontinuous. Membrane surfaces lacking a recognizable lamina densa were considered as lacking recognizable surface-associated ECM. Data was expressed as the mean and standard deviation of the mean.
Flow cytometry analysis
Cells maintained at different passage numbers were seeded 1 day before the
experiments in T75 flasks. For quantitative evaluation of the cell
surface-bound laminin, cells were incubated with 10 µg/ml of laminin-1 for
2 hours, washed with PBS and dissociated with cell dissociation buffer (Gibco
BRL, Rockville, MD). Cell concentration was adjusted to 5x105
cells per ml. Suspended cells were blocked with 5% goat serum in PBS, 0.5% BSA
and stained either with antilaminin or anti-receptors antibodies diluted in
washing buffer for 30 minutes. After washing with PBS, 0.5% BSA, 0.5% goat
serum cells were incubated with appropriate secondary antibodies conjugated
with FITC or Cy3 for 30 minutes. Following the last wash, cells were
resuspended in 500 µl of PBS containing 0.5% BSA, 0.1% paraformaldehyde and
analyzed by fluorescence-activated cell sorting (Epics-Profile II Coulter,
Beckman Coulter, Fullerton, CA). Control experiments without primary antibody
were performed in parallel.
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Results |
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Exogenous laminin-1 and laminin-2 each form two distinct
extracellular matrices on Schwann cell surfaces
To study Schwann cell-laminin interactions, we evaluated both mouse EHS
laminin-1 (1ß1
1) and human placental laminin-2/4
(
2ß1
1 and
2ß2
1). While
2-laminins
are major components found in developing and adult peripheral nerve basement
membranes, laminin-1 is closely related, possesses many shared properties, and
is more amenable to molecular dissection because of the availability of
perturbing reagents. These laminins bear three full-length short arms and have
been found to polymerize, interact with
6ß1,
6ß4 and
1ß1 integrins, and bind to dystroglycan.
Exogenous laminin-1 and laminins-2/4 were separately incubated with
cultured Schwann cells in DMEM/F12 culture medium for 4 to 24 hours at
37°C. The resulting laminin surface patterns were detected by
immunofluorescence microscopy following washing of cells to remove unbound
components and paraformaldehyde fixation of cells in the absence of detergent.
When Schwann cells were treated with exogenous laminin-1 (10 µg/ml) for 4
hours, extensive cell surface laminin coverage was detected by specific
immunofluorescence (Fig. 1a,b).
The pattern of staining changed little at longer incubation times (up to 24
hours). The level of endogenous laminin, as previously noted, was found to be
negligible compared with exogenous laminin in these experiments
(Fig. 1d). Schwann cells
incubated with 2 µg/ml of laminin-1 showed decreased cell surface
accumulation of laminin with two distinct patterns of distribution. One
pattern consisted of a dense plaque-like array referred to as the reticular
pattern because at high magnification small cleared areas were detected in an
otherwise confluent zone (Fig.
1e',f',e,f). This pattern was similar to the
laminin-induced honeycombed pattern observed to form on cultured myotubes in
the presence of exogenous laminin
(Colognato et al., 1999).
However, in Schwann cells the reticular mesh was denser with smaller
intervening empty spaces within the array. At higher concentrations (10-20
µg/ml), laminin covered a larger fraction of the cell surface area becoming
even denser with respect to size of the interstices of the array. The other
architecture (fibrillar) consisted of longer narrow linear structures with
limiting branching. (Fig. 1e,f,
arrows, and g). The reticular matrix pattern predominated in terms of surface
area coverage, particularly at higher laminin concentrations. Laminin-2/4
formed similar structures but had different concentration dependency.
Subsequent experiments focused on laminin-1 to permit evaluation with
domain-specific reagents.
|
Selective inhibition of reticular and fibrillar matrices
Laminin domain, integrin and dystroglycan contributions to the cell surface
accumulation of laminin-1 were evaluated
(Fig. 2) with laminin fragments
and receptor-blocking antibodies Ha2/5 (anti-ß1-integrin), and IIH6
(anti--dystroglycan). Laminin-1 fragments E4 (ß1 short arm domains
VI-V, 75 kDa), E1' (short arm complex excluding ß1 VI-IV, 450 kDa),
E8 (lower half of coiled-coil with LG1-3, 200 kDa), and E3 (distal G-domain LG
modules 4-5, 55 kDa) were incubated at 50 µg/ml each in the absence of
intact laminin for 4 hours with Schwann cells. Only fragment E3, which
possesses both dystroglycan and heparin binding sites that map to LG4
(Sung et al., 1997
;
Talts et al., 1999
), was
detected on the cell surface following incubation. The binding was punctate in
its appearance and less intense compared with intact laminin
(Fig. 2a,b).
|
Laminin fragments E3, E8, E4 and E1' (50 µg/ml each) were
incubated with laminin-1 (2 µg/ml) and examined by immunofluorescence
microscopy (Fig. 2). In
addition, random images collected from low-magnification (20x) fields
were analyzed to determine relative surface area coverage
(Fig. 2j). Treatment of cells
with ß1-integrin blocking antibody in the presence of laminin-1 prevented
formation of nearly all fibrillar laminin structures, leaving the reticular
structures largely intact (Fig.
2d). The addition of manganese, a divalent cation that enhances
ß1-integrin binding (Gailit and
Ruoslahti, 1988; Sonnenberg et
al., 1988
), increased the fraction of fibrillar matrix and
decreased the fraction of reticular matrix
(Fig. 2h). Fragment E3 had a
significant inhibitory effect on laminin accumulation (65%), while the other
fragments did not. The remaining bound laminin, following E3 inhibition, was
noted to be almost exclusively in a fibrillar or dot-like distribution
(Fig. 2f) (i.e. this fragment
prevented the formation of the reticular pattern). The effect of IIH6 antibody
was similar to E3 inhibition (Fig.
2e); however, overall, laminin inhibition was less (45%) and
occasional small surviving reticular patches were observed. When E3 and
anti-ß1 integrin antibody were incubated as a mixture with laminin,
overall laminin surface binding was decreased only slightly beyond that
achieved by E3 alone, however, both reticular and fibrillar patterns were
observed to be almost entirely absent, with remaining laminin distributed into
a punctate pattern (Fig. 2g).
Somewhat surprisingly, interfering effects were not appreciated with other
fragments at the concentrations used
(Colognato et al., 1999
). It
is possible that E8 (an integrin-binding fragment) may not have blocked
fibrillar anchorage due to integrin contributions arising from other domains,
while E1', which cannot directly interact with the cell surface, may not
have blocked matrix assembly because of a very high surface concentration of
laminin.
Contributions of laminin polymerization
Both 1- and
2-laminins polymerize to form network structures
that contribute a non-collagenous superstructure of basement membranes
containing these laminins (Cheng et al.,
1997
; Yurchenco and Cheng,
1993
; Yurchenco et al.,
1992
). Amino ethyl benzene sulfonyl fluoride (AEBSF) covalently
binds to the short arms of polymerizing laminin, selectively abolishing
polymerization without affecting cell adhesion, heparin binding, or
dystroglycan binding (Colognato et al.,
1999
). When Schwann cells were incubated with
non-polymerizing-laminin (both 5 µg/ml), the laminin was noted to still
bind to the cell surface (Fig.
3). However, the surface density was lower and more evenly
distributed, and the recognizable reticular architecture was greatly
diminished.
|
Topographical associations of laminin-1 with dystroglycan and
integrin receptors
Schwann cells, untreated or incubated for 8 hours with laminin-1 (2
µg/ml), were immunostained to detect both laminin and -dystroglycan
(Fig. 4). The pattern of
untreated cells was noted to be that of a dense and coarse punctate
distribution of dystroglycan epitopes across the entire exposed cell surface.
After laminin treatment, the pattern was now observed to be plaque-like,
consisting of more densely packed but finer dots, concentrated in zones of the
exposed cell surface. These dystroglycan zones co-localized with those
decorated by laminin in the reticular matrix. Following laminin incubation in
the presence of ß1-integrin-blocking antibody, the fibrillar component
was observed to be absent without affecting the reticular laminin
structures.
|
The integrin was found, in the absence of exogenous laminin, to be widely distributed as a fibrillar-like mesh (Fig. 5). Following an 8-hour treatment with laminin-1 the integrin pattern remained largely unchanged. Laminin within the fibrillar array was noted to correspond to a subset of the ß1-integrin fibrils [i.e. the laminin fibrils co-localized with only some of the integrin-staining fibrils (arrows)]. By contrast, no co-localization of reticular laminin patterns with integrin was identified. Although it is possible that the particular integrin fibrils that co-localized with laminin were induced to assemble in an otherwise pre-existing integrin array, the similarity of structures before and after laminin treatment were too great to be able to make such a distinction. In the presence of E3, which blocked formation of the reticular structure, the remaining fibrillar laminin co-distributed with a subset of integrin fibrils.
|
Effect of laminin matrix assembly on the distribution of cytoskeletal
proteins
To determine whether laminin-dependent matrix assembly induced a
rearrangement of cytoskeletal partners known to interact with dystroglycan and
integrins, we evaluated the distributions of utrophin, vinculin and paxillin
before and after treatment of Schwann cells with laminin
(Fig. 6). Utrophin, the
peripheral nerve homologue of dystrophin that binds to dystroglycan
(Yamada et al., 1996), was
distributed in a diffuse puncate pattern similar to that noted for
dystroglycan. Following overnight incubation with laminin, utrophin was noted
to be distributed into a more condensed pattern whose borders corresponded to
those of laminin reticular structures (Fig.
6b,c). Vinculin and paxillin are two cytoskeletal components found
in association with ß1-integrin in focal adhesions. These components were
not observed to be associated with either the reticular or fibrillar matrices
(Fig. 6d-g). Regardless of
laminin treatment, vinculin and paxillin were found to be present only with
the plasma membrane attached to the plastic surface (i.e. they were on a focal
plane below that of the two matrices) and were not altered in their apparent
distribution or number in response to added laminin.
|
Laminin-1 induction of an ultrastructural basement membrane
morphology
Schwann cells were incubated with laminin-1 (20 µg/ml) for 8 hours,
fixed and prepared for electron microscopy
(Fig. 7). The sections were cut
orthogonal to the plane of the plastic to enable visualization of the plasma
membrane that faced the media environment. In the absence of exogenous
laminin, this outer surface lacked discernable extracellular matrix
(Fig. 7a,C). Following
treatment with laminin-1, the outer plasma membrane surface was noted to be
covered by a linear extracellular matrix. This ECM appeared as a thin
continuous electron-dense line overlying a more lucent zone, measuring 20-40
nm in overall thickness (Fig.
7a,A). This ultrastructural morphology was that of a lamina
densa and lamina lucida, respectively, and was considered the
typical ultrastructural morphology of a thin basement membrane. Although long
stretches of basement membrane were detected, some sections revealed
discontinuities or portions of absent matrix as would be expected given less
than complete surface coverage by laminin as was shown by immunostaining of
the parallel cultures under same conditions
(Fig. 7a,B).
|
Blocking experiments with agents that interfere with laminin surface accumulation was performed in order to evaluate their effect on the appearance of an electron-dense extracellular matrix. After overnight incubation with 10 µg/ml of laminin-1 with or without blocking antibodies or fragments, cells were prepared for electron microscopy or for the control immunostaining. The lamina densa was judged to be continuous if it extended for a uninterrupted length of 2 µm or greater and discontinuous if it extended for less than that length (Fig. 7a). Formation of a lamina densa, either continuous or discontinuous, was prevented by a molar excess of fragment E3, a selective inhibitor of reticular matrix (Fig. 7b, left panel). By contrast, ß1-integrin blocking antibody, a selective inhibitor of fibrillar matrix, did not prevent formation of a continuous lamina densa. Thus there is a correlation between the reticular matrix and ultrastructural basement membrane morphology in which E3 inhibits the formation of both.
Role of laminin polymerization at the ultrastructural level
Treatment of Schwann cells with the non-polymerizing laminin
(AEBSF-treated) resulted in a substantially altered distribution and
significant reduction of laminin surface coverage by light immunomicroscopy as
previously noted (Fig. 3).
Electron microscopy was performed on cell samples incubated either with
laminin-1 or non-polymerizing laminin-1, both at 10 µg/ml for 18 hours. A
substantial decrease of a continuous lamina densa was noted with
partial shift to the discontinuous form
(Fig. 7b, graph).
Type IV collagen contributions
Basement membrane formed with laminin alone was considered to be a nascent
ECM in that it lacked type IV collagen, a major structural component of mature
basement membranes. No immuno-reactive type IV collagen was detected on
Schwann cell surfaces either before or after laminin treatment alone
(Fig. 8). However, if exogenous
type IV collagen was added to cultures, or if endogenous type IV collagen
secretion was promoted with L-ascorbic acid (which stabilizes collagen
triple-helix structure and which has been previously shown to cause collagen
deposition (Chernousov et al.,
1998)), collagen type IV was then observed to accumulate on cell
surfaces (Fig. 8 and data not
shown). This collagen was present either in fibrillar structures or small,
amorphous, brightly immunostaining particles. However, type IV collagen did
not accumulate into recognizable reticular patterns without added laminin. To
determine whether collagen type IV would associate with the reticular laminin,
the collagen (50 µg/ml) was incubated with laminin-1 (5 µg/ml) for 8
hours. The two proteins were observed to be co-localized in both the reticular
and fibrillar matrices.
|
When Schwann cells were incubated with type IV collagen in the presence of ß1 integrin-blocking antibody, it was no longer detected on the cell surface (Fig. 9). Thus, it appears likely that all type IV collagen surface accumulation is integrin-dependent in the absence of laminin. To study laminin-dependent component of collagen assembly we blocked ß1-integrin and examined type IV collagen accumulation in the presence of laminin (Fig. 9). Under these conditions, type IV collagen was detected only in laminin-containing reticular plaques. Thus, the nascent laminin-based basement membranes were competent to incorporate type IV collagen.
|
Schwann cell laminin-binding competency and expression of
dystroglycan and integrin as a function of cell passage
Schwann cell laminin-binding competency was found to be dependent on the
passage number. Low-passage cells (LPC; below 12) exhibited only a limited
coverage by laminin, nearly all in a fibrillar pattern, whereas high-passage
cells (HPC, >25) accumulated significantly higher amounts of laminin
predominately in the reticular pattern
(Fig. 10a,b). Schwann cells
retained expression of the differentiation marker S100 in both low and high
passage cells (Fig. 10c). FACS
analysis was used to evaluate the relative amount of laminin-1 (incubated at
10 µg/ml for 2 hours) that could bind Schwann cell surfaces
(Fig. 10d). Substantially
higher (about 200-fold) surface laminin-1 was noted in HPC compared with LPC
to the HPC. This result suggests that Schwann cells undergo a differentiation
during passaging that involves the expression of one or more critical laminin
cell surface anchors, enabling basement membrane formation.
|
To address this, the relative levels of cell surface ß1-integrin and
-dystroglycan on LPC and HPC were examined by flow cytometry
(Fig. 10e,f). Surprisingly,
the dystroglycan level remained essentially the same with ß1-integrin
only about twofold higher in the HPC. Such a difference in ß1-integrin
would not account for the greater than 100-fold overall laminin increase and
in any case is not expected to be relevant for basement membrane coverage.
Therefore the increased laminin accumulation on the surface of high passage
Schwann cells could not be explained simply by change in expression of these
laminin receptors. This suggests the existence of a third anchoring component
important for laminin assembly. Heparin/sulfatide binding is the other major
binding activity that maps to E3, the inhibiting fragment
(Talts et al., 1999
). A cell
surface heparan sulfate proteoglycan, or possibly a sulfatide, might
correspond to this anchor. Heparin (0.1 mg/ml), was found to eliminate most of
the laminin distributed in a reticular pattern (data not shown), supporting
this possibility.
![]() |
Discussion |
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Laminin and type IV collagen have been found to possess the intrinsic and
separate ability to polymerize, contributing the two major known architectural
networks of basement membranes (Yurchenco,
1994; Yurchenco et al.,
1992
; Yurchenco and Furthmayr,
1984
). However, these protein characteristics, in themselves, were
found to be insufficient to insure the formation of a cell-based ECM. An
additional critical factor appears to be that of cell anchorage. For such
assembly to occur on Schwann cells, the cells had to be in a competent state
for laminin binding. Low passage cells bound laminin to a very limited degree
with such laminin detected in fibrillar-like arrays but not nascent basement
membranes. By contrast, cells at later passage became capable of binding
laminin with over a 100-fold higher capacity. Addition of laminin to these
cells initiated assembly of nascent basement membranes that predominated over
the fibrillar matrix in terms of surface coverage. Nascent basement membrane,
enabled by competency and triggered with exogenous laminin, required surface
binding through laminin LG modules 4-5 located in the large globular domain at
the end of the long arm. It seems likely that it is this binding interaction
that is rendered permissive through cell-passaging. In comparing incompetent
and competent laminin-binding cells, no difference was detected in cell
surface dystroglycan and little difference was detected in ß1-integrin.
This surprising finding suggests that an additional cell surface anchor for
LG4-5 is either expressed or activated as the cell becomes competent. Given
that heparin/heparan sulfate binding is the other major activity identified in
E3 (mediated by LG module-4), surface anchorage may depend upon a cell
surface-associated heparan sulfate proteoglycan (HSPG). Consistent with this
consideration, we have found that heparin inhibits the accumulation of both
laminin-1 and laminins-2/4 into nascent basement membrane. Although
dystroglycan binding is in itself inhibited by heparin in the case of
laminin-1, this is not the case with laminin-2
(Pall et al., 1996
) and
therefore not likely to be dependent on dystroglycan alone. It is also
possible that an anchoring contribution could be provided by sulfated
glycolipid (sulfatide) that bind to the heparin sequences
(Roberts et al., 1985
;
Talts et al., 1999
). The
putative surface anchor, if a HSPG, is likely to be linked to the dystroglycan
interaction, perhaps acting as a `co-receptor', given the finding that
dystroglycan and heparin binding sequences are located on the same LG-4
protein face (Tisi et al.,
2000
). We suggest that the phenotypic switch thought to occur in
Schwann cells upon neuronal contact may involve this anchoring state
passage-dependent transition, a requirement over and above a separate
requirement for the initiation of the synthesis of basement membrane
components. Given that Schwann cells undergo a developmental transition from
individual cells to ones that envelop axons and produce an abaxonal basement
membrane, what might be the functional role of the fibrillar matrix? Its
structural topography is similar to that of the integrin-associated fibrillar
architecture induced by fibronectin on fibroblasts, cells that cannot form
basement membranes (Klass et al.,
2000
; Zhong et al.,
1998
). The fibrillar matrix in Schwann cells might have a function
important for migration and neuronal growth-cone pathfinding in the stages
that precede envelopment and that occurs both in nerve development and
regeneration.
The characteristics of Schwann cell basement membrane assembly were related
to those observed on myotubes (Colognato
et al., 1999) in that both showed a selective E3-dependency and
were inhibited with dystroglycan blocking antibody, but not by integrin
blocking antibody. Both also showed a dependency on laminin polymerization
with the greatest contribution seen in myotubes. A substantial difference was
seen in the co-localization of laminin with integrin and vinculin in the case
of myotubes, and its lack in Schwann cells. Thus, the receptor and
cytoskeletal partners associated with laminin can differ under different
circumstances, suggesting that they could be found to be cell-type specific.
In Schwann cells, the fibrillar matrix was not associated with an
ultrastructurally defined basement membrane, although it did associate with
type IV collagen. Therefore, this distinct ECM, discontinuous in its
distribution, appears to be a unique entity. Similar structures have been
identified on Schwann cells previously, and our data are in agreement with the
conclusion that these ECMs are not precursor to basement membrane
(Chernousov and Carey, 2000
;
Chernousov et al., 1998
). Given
the ability of basement membrane-incompetent Schwann cells to assemble these
matrices, and given their resemblance to fibrillar matrices seen on
fibroblasts, these ECMs might be primarily attributes of a variety of
migrating cells that are not involved in the process of basement membrane
assembly.
The factors that determine which cell surfaces can host basement membranes have been unexplained in the past. Muscle and fat cells become enveloped by these ECMs while epithelial cells develop a basement membrane restricted to their abluminal surface. Fibroblasts, even though they secrete basement membrane components, do not assemble their own basement membranes. The data from this study argue for the existence of a laminin G-domain interacting, integrin-independent anchoring complex that imparts competency for basement membrane assembly and that is to be distinguished from receptors that mediate fibrillar matrix. Future goals will be to elucidate cell surface molecules and their cytoskeletal partners that constitute the complex and to determine the mechanisms that differentially drive ECM assembly and mediate functions.
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