1 MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and
Molecular Biology, University College London, London, WC1E 6BT, UK
2 Department of Medicine, University of Wisconsin, Madison, WI 53706, USA
Present address: Department of Cell Biology, NC1-110, Lerner Research
Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH
44195, USA
* Author for correspondence (e-mail: adamsj{at}ccf.org )
Accepted 12 March 2002
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Summary |
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Key words: Cell adhesion, Extracellular matrix, Oligomerisation, Actin cytoskeleton, Fascin
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Introduction |
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Within the TSP family, TSP-1 and TSP-2 are most closely related in terms of
their domain organisation and degree of sequence identity
(O'Rourke et al., 1992;
Laherty et al., 1992
). TSP-1
and TSP-2 form a distinct subgroup, subgroup A, because they are the only
members of the family to contain a procollagen homology region and TSP type 1
repeats and to be assembled as trimers
(Adams and Lawler, 1993
). TSP-1
and TSP-2 transcripts and proteins have distinct patterns of expression, most
probably because of different mechanisms of transcriptional regulation
(Bornstein et al., 1991
;
Bornstein, 1992
;
O'Rourke et al., 1992
;
Iruela-Arispe et al., 1993
;
Tucker 1993
;
Tooney et al., 1998
).
TSP-1-null mice have decreased embryonic viability, are born with lordotic
curvature of the spine and have increased numbers of circulating white blood
cells. In addition, as neonates they develop chronic inflammation in the lung
that leads to their death by pneumonia
(Lawler et al., 1998
).
TSP-2-null mice have connective tissue abnormalities associated with
disorganisation of collagen fibrils, increased bone density, increased
vascular density in many tissues and a bleeding diathesis
(Kyriakides et al., 1998
).
This multiplicity of phenotypes emphasises the general roles of TSP-1 and
TSP-2 in tissue organisation.
The available mechanistic evidence indicates that TSP-1 and TSP-2 may have
overlapping effects on cell function in the different tissues in which they
are expressed. Deletion of either gene leads to perturbation of skin wound
healing and wound healing angiogenesis, and overexpression of TSP-1 suppresses
wound healing (Lawler et al.,
1998; Kyriakides et al.,
1999
; Streit et al.,
2000
). In cell culture assays, TSP-1 and TSP-2 both act as
adhesion molecules and are antiangiogenic
(Good et al., 1990
;
Chen et al., 1994
;
Volpert et al., 1995
). TSP-1
also induces cell migration (reviewed by
Adams et al., 1995
). Peptides
derived from the N-terminal domains of TSP-1 and TSP-2 promote disassembly of
focal adhesions when added to preadherent cells
(Murphy-Ullrich et al., 1993
).
The mechanisms by which TSP-1 binds to cells have been studied intensively and
involve interactions with cell-surface proteoglycans, integrins, CD47 and the
LDL-receptor-related protein, LRP (for reviews, see
Lawler, 2000
;
Adams, 2001
). For the limited
number of cell types that have been examined, TSP-2 was also found to bind to
cells through multiple contacts with integrin and proteoglycan adhesion
receptors and LRP (Chen et al.,
1994
; Chen et al.,
1996
) (reviewed by Bornstein
et al., 2000
).
Cell adhesion to TSP-1 is characterised by a unique cytoskeletal
organisation in which cells form lamellae with radial spikes and ribs, which
contain F-actin and the actin-bundling protein fascin
(Adams, 1995;
Adams, 1997
). These structures
are also needed for cell migration on TSP-1
(Adams 1997
;
Adams and Schwartz, 2000
).
Induction of spikes by TSP-1 is transduced by syndecan-1 and requires the
syndecan-1 core protein and glycosaminoglycan substitutions at residues S45
and S47 (Adams et al., 2001
).
Skeletal myoblasts form particularly large arrays of spikes and attach to
TSP-1 by binding to the type 1 repeats and C-terminal globular domains
(Adams, 1995
;
Adams and Lawler, 1994
).
However, a monomeric C-terminal domain shows low cell attachment activity and
does not support full cell spreading or spike formation
(Adams and Lawler, 1994
)
(J.C.A., unpublished). Thus the mimimal requirements for induction of cell
spreading and fascin spikes by TSP-1 are not known. Furthermore, TSP-2-null
fibroblasts show impaired spreading on rigid culture substrata, but the direct
effects of TSP-2 on the cytoskeleton are not known
(Kyriakides et al., 1998
).
To date, the mechanisms of action of TSP-1 and TSP-2 have principally been
determined by the use of short, linear peptides that correspond to
cell-binding sites, or reagents that block particular adhesion receptors, both
of which act as inhibitors of cell attachment to intact TSP1 or TSP-2. Many
studies have demonstrated functional effects of peptides derived from TSP-1 on
cell adhesion and migration (e.g. Guo et
al., 1992; Wang and Frazier,
1998
; Iruela-Arispe et al.,
1999
). However, the interpretation of these data is complicated by
uncertainties as to the positioning of the peptide motifs within the natural
tertiary structures of TSP modules. Preparation of recombinant protein units
from TSPs has proved challenging because of the large size of these molecules
and the need for validation of the physical structure of an engineered unit
against that of the native protein. Although native TSP-1 is trimeric,
fragments and monomeric portions are reported to occur in vivo
(Rabhi-Sabile et al., 1996
;
Bonnefoy and Legrand, 2000
),
and it is not clear whether these moieties also have biologically relevant
effects on cell adhesion and migration. To address the important question of
the mechanisms by which TSP-1 or TSP-2 use to induce fascin spike cytoskeletal
structure, we have prepared a panel of recombinant, physically characterised
monomeric or trimeric protein units of TSP-1 and TSP-2. We demonstrate that
although certain monomeric protein units have cell-attachment activity,
trimeric assembly of the type 3 repeats and C-terminal globule is needed to
achieve cell spreading and fascin spike cytoskeletal organisation. The
implications of these novel results for the design of TSP-containing matrices
for biomedical or tissue engineering applications are discussed.
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Materials and Methods |
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Preparation of monomeric and trimeric TSP recombinant protein
units
Constructs containing portions of the coding sequences of human TSP-1 or
human TSP-2 were prepared in the plasmid pAcGP67.coco (COCO). The COCO vector
is the baculovirus transfer vector pAcGP67A (Pharmingen) modified by the
addition of DNA to encode a short linker (AAG) and a six His-tag (HHHHHH)
3' to the cloning site, with or without an intervening thrombin cleavage
site (LVPRGS) (Misenheimer et al.,
2000). The cDNA inserts were generated by PCR of appropriate
segments of the cDNAs for human TSP-1 or TSP-2 to yield the modules shown in
Fig. 1. The inserts were
ligated into the COCO vector using standard molecular biology techniques
(Sambrook et al., 1993
). In
the N-terminus of each construct the signal sequence from the acidic
glycoprotein gp67 of the AcNPV virus directly precedes the cloning site and
targets the protein for secretion. The thrombin cleavage site allows the
His-tag to be removed after purification of recombinant proteins that are not
themselves susceptible to thrombin cleavage. Recombinant baculoviruses were
obtained from Sf9 insect cells (Invitrogen, Netherlands) by cotransfection of
the COCO constructs with Baculogold DNA (Pharmingen) according to standard
procedures. Individual recombinant virus clones were isolated by plaque
purification. After three rounds of viral amplification in Sf9 cells, pass
three virus strains were made and tested for protein production by a TSP
immunoblot. The highest producing clones were chosen for subsequent use.
Serumfree suspension cultures of High Five (Invitrogen) or Sf9 insect cells
were infected with pass three virus at multiplicities of infection of two to
10, depending on the construct and its exact protein yield. The conditioned
media was harvested between 49 hours and 72 hours post-infection, after which
the His-tagged recombinant proteins were purified by nickel-chelation
chromatography (matrix from Invitrogen) as described
(Meisenheimer et al., 2000
).
The trimeric or monomeric status of proteins and their purity was assessed by
Coomassie blue staining of SDS-polyacrylamide gels run under reducing or
nonreducing conditions in conjunction with immunoblotting with antibodies
specific to TSP-1 or TSP-2. Protein concentrations were quantified by the
Bradford assay (Bradford,
1976
) using bovine serum albumin as a standard (BioRad kit). The
preparations were stored in portions in TBS containing 0.3 mM CaCl2
at -80°C.
|
Cell attachment assay
Flat-bottomed 96-well polystyrene plates (Nunc) were coated with 50 nM
human plasma FN, 50 nM recombinant TSP-1 or between 50 nM and 1 µM of the
TSP recombinant protein units for 16 hours at 4°C. The efficiency of
protein coating was confirmed in two ways: by determining the TSP content of
SDS-PAGE sample buffer extracts of the proteins from dishes after coating by
gel electrophoresis and Coomassie blue staining or immunoblotting using
standard procedures (Adams et al.,
1999), and by comparison of the protein concentration of the
coating solution before and after the adsorption period using the Bradford
method (BioRad kit). By both measurements, 80% to 90% of each protein became
adsorbed. Protein-coated surfaces were washed twice with Tris-buffered saline
(TBS) containing 2 mM CaCl2 and blocked with 1 mg/ml of
heatdenatured bovine serum albumin (BSA, Sigma) for 1 hour at 23°C. C2C12
myoblasts or rat aortic VSM cells were trypsinised, washed and suspended at
concentrations of 5x105 cells/ml, and a total of 50,000 cells
were added per well and incubated for 1 hour or for other time periods at
37°C. Non-adherent cells were removed by gentle rinsing, and the number of
adherent cells was quantified by measurement of cellular phosphatase activity
(Prater et al., 1991
).
Briefly, the cells were incubated for 90 minutes at 37°C with 100 µl of
substrate lysis solution (1% Triton X-100 containing 6 mg/ml p-nitrophenyl
phosphate in 50 mM sodium acetate buffer, pH 5). The reaction was stopped by
addition of 50 µl of 1 M NaOH, and the optical density was measured at 410
nm in a microplate reader.
In experiments to determine the mechanisms of cell attachment, 5 µg/ml
of anti-integrin antibodies, 1 mM GRGDSP peptide, 100 µg/ml heparin, 300
µg/ml chondroitin sulphate A or 1 mM of a synthetic peptide,
KRFYVVMWKQVTQS, which corresponds to the CD47-binding motif within the
C-terminal globule of TSP-1 (Gao et al.,
1996), were added to the cell suspensions at the time of plating.
The concentrations used were based on the known sensitivities of other cell
types to these reagents and, in the case of the anti-integrin reagents, the
maximum levels of inhibition obtainable in pilot titration experiments against
cell attachment to their known major matrix ligands. The number of attached
cells was quantified as described above. Each experimental condition was
carried out in triplicate in a single experiment, and data from three
independent experiments were collated for descriptive statistical analysis in
Excel worksheets.
Immunofluorescent staining of cells for F-Actin and fascin
Glass coverslips were coated with 50-100 nM FN, VN or recombinant TSP-1, or
varying concentrations of the TSP-1 or TSP-2 recombinant protein units for 16
hours at 4°C. The surfaces were blocked, and the cells were prepared for
attachment assay as described above. Non-adherent cells were removed by gentle
rinsing in TBS. For staining of F-actin, cells were fixed in 3.7%
paraformaldehyde for 10 minutes, washed briefly with TBS, permeabilized with
O'Neill buffer (O'Neill et al.,
1990) and washed again in TBS. Permeabilised cells were incubated
with tetramethylrhodamine-conjugated phalloidin (Sigma) for 90 minutes, washed
and mounted on slides using Vectashield mounting medium (Vector Laboratories
Inc, CA).
For fascin staining, cells were fixed in absolute methanol for 10 minutes, washed with TBS and incubated with 55k2 antibody to fascin for 90 minutes. Cells were washed and incubated with FITC-conjugated anti-mouse IgG (Sigma) for 60 minutes, then washed, mounted and observed under an Axioskop epifluorescence microscope (Zeiss, Germany), and photographs were taken on Kodak T-Max 100 film. In other experiments, digital images were captured by a Hammamatsu C5985 CCD camera controlled by Improvision Openlab 2.2.5 software and processed into Adobe Photoshop 5.5 for the montages presented here.
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Results |
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The proteins that lacked the N-terminal domain, N, and included the
oligomerisation domain with the entire C-terminal portion of the subunits
(designated DelN(1) and DelN(2), Fig.
1) were also secreted as stable trimers that resolved on SDS-PAGE
gels with apparent molecular masses of 305 kDa under non-reducing conditions
and 103 kDa under reducing conditions (Fig.
2B). Detailed physical analysis of the structure of the protein
units using resistance to proteolytic digestion, circular dichroism and
fluorescence spectroscopy showed that their physical properties were similar
to those of intact TSP-1 and TSP-1 fragments derived by limited proteolysis
(Panetti et al., 1999;
Misenheimer et al., 2000
)
(D.F.M., unpublished). The physical structure of the N-terminal globular
domain does not depend on trimerisation
(Misenheimer et al., 2000
).
The recombinant protein units are thus a complete, defined and structurally
appropriate set of reagents with which to probe specific functional properties
of TSP domains.
Cell attachment activity is contained within the Type 1 repeats and
Type 3 repeat/C-terminal globule recombinant protein units
We first compared the ability of the TSP recombinant protein units to
support cell attachment. To ensure that all of the protein activities observed
were of general relevance, the assays were carried out on two non-transformed
cell types that spread on intact TSP-1:C2C12 myoblastic cells, which were
derived from skeletal muscle where TSP-1 and TSP-2 are co-expressed, and rat
aortic vascular smooth muscle cells (VSM cells), derived from arterial smooth
muscle where TSP-1 is implicated in the migratory phenotype
(Majack et al., 1988;
Tucker, 1993
;
Iruela-Arispe et al., 1993
;
Tooney et al., 1998
;
Wang and Frazier, 1998
). In
pilot experiments we found that the monomeric TSP-1 or TSP-2 recombinant
protein units, coated at a concentration of 50 nM at which intact TSP-1 or
fibronectin (FN) very effectively support cell attachment
(Fig. 3A), did not promote cell
attachment of either C2C12 myoblasts or VSM cells. We established in control
experiments that all the recombinant protein units were adsorbed efficiently
to plastic or glass surfaces. A titration experiment using up to 2 µM
coating concentration of each protein was carried out to establish whether
higher concentrations of the TSP units promote cell attachment. From these
experiments, 1 µM coating concentrations for the E3Ca(1) and CP123(1) units
from TSP-1 were found to support the attachment of both cell types, with
half-maximal attachment occurring at a coating concentration of 500 nM (data
not shown). Pilot time-course experiments showed that maximal cell attachment
was reached at incubation times of 45 minutes or longer (data not shown).
Therefore, the subsequent experiments used 1 µM coating concentrations for
the recombinant protein units and a 1 hour time point.
|
We next quantified the attachment activities of recombinant protein units that contained different structural domains at 1 µM to 2 µM coating concentrations and compared their activities with those of intact TSP-1 and intact FN at 50 nM. Intact TSP-1 promoted approximately 90% attachment of C2C12 myoblasts and VSM cells compared with cell attachment to intact FN (Fig. 3A). Of the units from TSP-1, DelNo(1) promoted 70% to 80% cell attachment (Fig. 3A). The trimeric N-terminal unit, NoC(1), supported attachment of around 10% of the cells, which was not significantly different from background attachment to BSA (Fig. 3A). CP123(1) promoted 50% attachment of skeletal myoblasts and nearly 45% attachment of VSM cells. E3Ca(1) promoted 36% attachment of C2C12 cells and 47% attachment of VSM cells. Of the units of TSP-2, DelNo(2) supported around 70% attachment of both cell types relative to FN, and thus had similar quantitative attachment activity to DelNo(1). Cell attachment to the NoC(2) trimeric unit was not above background (Fig. 3A). E3Ca(2) had almost identical attachment activity to E3Ca(1) for both cell types (data not shown). The number of attached cells did not increase further when 2 µM coating concentrations of the recombinant protein units were used, therefore we conclude that the values obtained represent maximal levels of cell attachment to the recombinant protein units. We also tested 50:50 mixtures of 2 µM DelNo(1) with 2 µM NoC(1), or 2 µM DelNo(2) with 2 µM NoC(2), to examine whether reconstitution of all the domains of a subgroup A TSP subunit in trans would promote higher cell attachment than the individual units. Cell attachment to these mixtures was indistinguishable from attachment to DelNo(1) or DelNo(2) coated alone at 1 µM (data not shown).
To control for the possibility that the protein conformation of the
monomeric recombinant protein units was not optimal for cell attachment
activity, we performed cell attachment assays with the TSP modules coated in
sodium acetate buffer at pH 4. An acidic pH has been shown to improve the
adsorption of TSP-1 to tissue culture plastic
(Kaesberg et al., 1989). Under
these conditions, the percentage attachment of both C2C12 cells and VSM cells
was identical to that obtained on the protein units coated in the standard
buffer conditions (data not shown). We also extended the attachment assays to
90 minutes or 120 minutes to examine whether longer incubation times were
needed to detect cell attachment to certain recombinant protein units. We did
not detect any significant quantitative increase in the percentage of cells
attached or any change in the morphology of attached cells at longer
incubation times on any of the TSP units and therefore continued to use the 60
minute incubation period in further experiments (data not shown). Overall,
these experiments show that the major cell attachment sites of subgroup A TSPs
are located in the type 1 repeats and type 3 repeats/C-terminal globule. The
N-terminal domain and type 2 repeats are essentially non-adhesive. It was of
note that the monomeric recombinant protein units that supported cell
attachment had 10- to 20-fold lower activity on a molar basis than intact
TSP-1.
The mechanism of attachment of VSM cells depends on multiple adhesion
receptors
The attachment mechanism of C2C12 myoblasts to TSP-1 was studied previously
using domain-specific antibodies and various attachment inhibitors. C2C12
cells interact with the type 1 repeats of TSP-1 and the C-terminal globular
domain in a proteoglycan-dependent process that shows little or no sensitivity
to anti-integrin reagents (Adams et al.,
1998; Adams et al.,
2001
). We obtained identical results for the mechanisms of C2C12
attachment to DelNo(1) and DelN(1) (data not shown). To characterise the
attachment mechanisms of VSM cells, specific inhibitors of candidate
cell-matrix adhesion receptors were first tested for perturbation of cell
attachment to DelNo(1), the monomer that showed maximal cell-attachment
activity (Fig. 3A). Neither
GRGDSP peptide nor a function-blocking antibody to the rat ß3 integrin
subunit blocked attachment, even when tested at a range of concentrations
that, at their maximum, reduced cell attachment to VN by 75%
(Fig. 3B) (data not shown). In
contrast, a function-blocking monoclonal antibody to rat ß1 integrin
subunit maximally inhibited attachment by 32%. The same concentration of this
reagent blocked VSM cell attachment to FN by 92%
(Fig. 3B) (data not shown). We
also tested a function-blocking polyclonal antibody to human ß1 integrin
subunit that has broad species cross-reactivity
(Adams et al., 1998
). At a
concentration that maximally inhibited attachment to FN by 95%, this reagent
maximally inhibited VSM attachment to DelNo(1) by 41%, and no further increase
in inhibition was produced by the use of a two-fold higher concentration (data
not shown).
Several ß1 integrins have been implicated in the attachment of
individual cell types to TSP-1 (Yabkowitz
et al., 1993; Wang and
Frazier, 1998
; Krutsch et al., 1999). We therefore tested the
effects of the available function-blocking antibodies to rat integrin
subunits (Mendrick and Kelly,
1993
). Antibodies to the
4 or
5 subunits did not
inhibit attachment to DelNo(1) at any of the concentrations tested
(Fig. 3B) (data not shown),
whereas the antibody to the
2 subunit maximally inhibited attachment by
42%, which is equivalent to the level of inhibition obtained with the
anti-ß1 subunit reagents (Fig.
3B). This
2 integrin antibody maximally inhibited VSM cell
attachment to collagen IV by 60% (data not shown). Proteoglycans are important
in cellular responses to TSP-1 (e.g. Sun
et al., 1989
; Mikhailenko et
al., 1995
; Chen et al.,
1996
; Shafiee et al.,
2000
; Adams et al.,
2001
; Li et al.,
2001
), and both heparin and chondroitin sulphate A caused
approximately 25% inhibition of cell attachment to DelNo(1)
(Fig. 3B). The CD47-binding
peptide reduced cell attachment by 47%
(Fig. 3B).
Since none of the inhibitory reagents blocked cell attachment completely,
it appeared likely that multiple adhesion receptors were involved in VSM cell
attachment to DelNo(1). We therefore proceeded to test the reagents in
combination. The levels of inhibition produced by pairwise combinations of the
inhibitors were not significantly different from those achieved using the
single reagents (data not shown). In contrast, the triple combination of the
antibody to ß1 integrin subunit, heparin and the CD47-binding peptide
reduced the attachment of VSM cells to DelNo(1) by 80%, a significant increase
in inhibition compared with the double combinations (P=0.002 relative
to heparin+CD47 peptide, or ß1 antibody+heparin, P=0.01 relative
to ß1+CD47 peptide; Fig.
3B). These results indicate that 2ß1 integrin,
proteoglycans and CD47 act in combination to support VSM cell attachment to
DelNo(1).
Cell-attachment activity depends on trimeric assembly of active
domains
Whereas 50-100 nM coating concentrations of intact TSP-1 were sufficient to
achieve maximal quantitative cell attachment at a level comparable to that of
FN (Adams et al., 1998) (this
study), 500 nM to 1 µM coating concentrations of the CP123, E3Ca or DelNo
units were needed to bring about maximal cell attachment
(Fig. 3A). This led us to
consider the effect of subunit trimerisation on cell attachment activity. We
compared the attachment activity of 100 nM TSP-1 with 100 nM of the monomeric
and trimeric versions of the CP123 and DelN recombinant protein units. For
both cell types, the trimeric DelN(1) and DelN(2) proteins showed comparable
activity to intact TSP-1, and the trimeric oCP123(1) had 50% attachment
activity, whereas at the 100 nM concentration the monomers were non-adhesive
(Fig. 4A, shown for VSM cells
only). The mechanisms of VSM cell attachment to the trimeric DelN(1) and
DelN(2) proteins paralleled attachment to DelNo(1) in that maximal inhibition
of attachment required the combination of anti-
2ß1 integrin,
heparin and CD47 peptide (Fig.
4B). Cell attachment to oCP123 was maximally blocked by a
combination of anti-
2ß1 integrin and heparin. A previously
described recombinant protein corresponding to the procollagen region alone
(Misenheimer et al., 2000
) had
negligible attachment activity, indicating that the binding site for
2ß1 integrin is located in the type 1 repeats (data not shown). In
accordance with the mapping of the CD47-binding site to the C-terminal globule
of TSP-1, the CD47 peptide, alone or in combination, did not affect cell
attachment to oCP123 (Fig. 4B)
(Gao et al., 1996
). Thus, cell
attachment to large monomeric or trimeric units from TSP-1 depends on similar
mechanisms; the mechanisms of VSM cell attachment to DelN(1) and DelN(2) are
similar and in each case the avidity or affinity of interaction is higher on
the trimerised modules than on equivalent monomers.
|
Trimerisation of the TSP C-terminal region is needed for induction of
cell spreading and fascin spike organisation
The attachment of C2C12 myoblasts or VSM cells to intact TSP-1 results in
cell spreading and assembly of fascin spikes, which are also needed in cell
migration on TSP-1 (Adams,
1997; Adams and Schwartz,
2000
) (J.C.A., unpublished). To determine the effects of the
recombinant protein units on fascin spike formation, we first examined cell
spreading and cytoskeletal organisation in cells attached to monomeric
DelNo(1) or DelNo(2) used at concentrations that showed similar
cell-attachment activity to 100 nM TSP-1
(Fig. 3). Both cell types
spread on FN, assembled actin microfilament bundles and had a diffuse
distribution of fascin (Fig.
5). However, neither DelNo(1) nor DelNo(2) supported effective
cell spreading of either cell type. The cells on DelNo(1) and DelNo(2) were
rounded with an irregular cell periphery or, in some cells within the
population, small marginal actin ruffles. Actin microfilament bundles were not
assembled, fascin distribution was diffuse and no fascin-containing structures
were formed at cell margins (Fig.
5). Vinculin distribution was also diffuse (data not shown). We
also examined F-actin and fascin organisation in cells on CP123(1) or E3Ca(1).
On CP123(1), cells remained round. On E3Ca(1), cell margins were more
irregular but the extent of spreading was no more than on the DelNo(1) units,
and fascin spikes were not apparent (data not shown).
|
We also examined spreading and cytoskeletal organisation of C2C12 cells and VSM cells plated on a 50:50 equimolar mixed matrix of DelNo(1) and NoC(1), or DelNo(2) and NoC(2). The mixtures did not support spreading or cytoskeletal organisation of either cell type (data not shown). We tested whether NoC(1) or NoC(2) in soluble form could affect F-actin organisation in C2C12 myoblasts or VSM cells adherent on FN. No reproducible effects on cytoskeletal organisation were apparent in these cell types (data not shown). Thus, even under conditions of maximal cell attachment, the DelNo units do not induce cell spreading, F-actin cytoskeletal organisation or assembly of matrix contacts.
We proceeded to test the capacity of the trimeric adhesive molecules to induce cytoskeletal organisation. Strikingly, both DelN(1) and DelN(2) supported cell spreading to a level comparable with that documented on intact TSP-1 when tested at equivalent molarity. On either module, C2C12 and VSM cells adopted angular, protrusive morphologies with multiple lamellipodial regions enriched in F-actin. The assembly of fascin spikes and ribs was clearly apparent in both cell types on DelN(1) (Fig. 5). C2C12 cells adherent with DelN(2) also assembled arrays of fascin spikes; however in VSM cells the organisation of fascin into radial spikes was less pronounced. The cells tended to form marginal ruffles, in which fascin was enriched at the cell peripheries but was not tightly organised into bundles like those seen in cells on DelN(1) (Fig. 5). In contrast, the trimeric oCP123(2) protein did not support cell spreading or induce fascin spikes (Fig. 5). These results demonstrate that monomers and trimers that include the C-terminal type 3 repeats/C-terminal globule show a fundamental, concentration-independent distinction in activity for the induction of cell spreading and cytoskeletal organisation.
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Discussion |
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Little is known about the adhesive effects of TSP-2, and our data provide new insights into its effects on the cytoskeleton. The matched recombinant reagents enabled direct comparisons to be made between the properties and mechanisms of action of TSP-1 and TSP-2. For both molecules, cell-attachment activity is located in the CP123 and E3Ca units and maximal cell-attachment activity is supported by these domains in combination. Both DelN(1) and DelN(2) supported cell spreading and actin cytoskeletal organisation and had a similar potency to intact TSP-1. A direct comparison with intact TSP-2 could not be made because of poor production of the recombinant protein.
Multimerisation of other ECM molecules has been documented to affect other
aspects of cell function. The proliferation and migration of vascular smooth
muscle cells was inhibited on polymerised collagen I
(Koyama et al., 1996;
Ichii et al., 2001
), whereas
the presence of proteolytic fragments of collagen I stimulated cell rounding
and focal adhesion disassembly (Carragher
et al., 1999
). Lymphocytes require activation signals in order to
attach to monomeric FN and fibrinogen, yet they attached and initiated
intracellular signals without activation on polymerised forms of these matrix
molecules (Stupack et al.,
1999
). Experiments in which the same mean concentration of RGD
peptide is presented to cells in clusters or as a uniform sheet have directly
demonstrated that clusters of ligand are more effective at inducing cell
spreading and motility than monomers
(Maheshwari et al., 2000
). In
the case of TSPs, our data provide the first direct demonstration of a
necessity for TSP trimer assembly for the induction of fascin spikes. This
accords with the recent demonstration that antibody-mediated clustering of
syndecan-1 is sufficient to induce bundling of F-actin and fascin in spikes
(Adams et al., 2001
).
The trimerisation of subunits is likely to be required for a number of the
functions attributed to TSP-1 or TSP-2 that depend on cell spreading and thus
on the presence of an organised actin cytoskeleton. These activities include
the regulation of cell-cell interactions and cell migration during wound
healing or the motility of smooth muscle cells in atherosclerotic lesions
(reviewed by Adams, 2001). Our
results also raise the possibility that monomeric TSP moieties that have been
documented in tissues (Bonnefoy and
Legrand, 2000
) could have different effects on cell adhesion
behaviour by supporting cell attachment and not cell spreading.
In summary, our results define for the first time activities of TSP cell-attachment sites across a set of appropriately structured recombinant protein units. The data reveal major similarities in the effects on the actin cytoskeleton between TSP-1 and TSP-2. The striking differences in spreading and cytoskeletal organisation obtained on monomeric or trimeric C-terminal protein units demonstrate higher order aspects of TSP function that have implications for the molecular mechanisms of action of TSP-1 and TSP-2 in cell locomotion and tissue-modelling cell interactions. These aspects of function would need to be taken into account when designing potential preventative or therapeutic strategies or producing engineered ECM to regulate and mimic biological effects of TSP-1 and TSP-2 in wound healing, tumour angiogenesis and vascular function.
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