1 Howard Hughes Medical Institute and
2 Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005-1892, USA
*Author for correspondence (e-mail: richard{at}bioc.rice.edu)
Accepted 15 May 2002
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SUMMARY |
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Key words: Counting factor, Cell population size, Dictyostelium discoideum
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INTRODUCTION |
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Using shotgun antisense transformation for mutagenesis, we found a Dictyostelium transformant we named smlAas that formed very small groups and fruiting bodies (Spann et al., 1996). The smlAas cells formed streams with normal morphology and size which then broke up into a large number of small groups. Disrupting the smlA gene using homologous recombination resulted in cells with a phenotype identical to that of smlAas (Spann et al., 1996
). SmlA is a cytosolic protein with no strong similarity to any known protein. It appears to regulate some aspect of secretion, as the smlA and smlAas cells secrete a soluble factor which when added to early developing wild-type cells causes them to form small groups (Brock et al., 1996
).
One way in which cells can theoretically sense the size of a tissue or group of cells is if all the cells in the group secrete a diffusible factor. As the number of cells in the group increases, the concentration of the factor will increase, and thus the cells themselves or a different set of cells can sense the size of the group (Brock et al., 1996; Clarke and Gomer, 1995
; Gomer, 2001
). We hypothesized that the factor secreted by the smlA cells might be an example of such a factor. Using conventional protein chromatography and the ability of this factor to cause wild-type cells to form small groups as a bioassay, we partially purified the factor and found that it appeared to be a
450 kDa complex of polypeptides we named counting factor (CF). We found that CF was also secreted by wild-type cells, although to a lesser extent than by smlA cells. This suggested that the smlA cells oversecrete a factor that the wild-type cells use to sense the number of cells in a group, so that when there is a small number of smlA cells in a group, the higher concentration of CF causes these cells to sense that there is a much larger number of cells (Brock and Gomer, 1999
).
A SDS-polyacrylamide gel of CF showed that there are at least 5 different polypeptides in the complex (Brock and Gomer, 1999). We obtained N-terminal amino acid sequence for 3 of the proteins; another 2 were blocked. We isolated the cDNA encoding a 40 kDa component, which we named countin. The derived amino acid sequence of the countin protein has some similarity to amoebapores, a superfamily of proteins that bind to membranes (Zhai and Saier, 2000
). A prediction of the secreted factor being used to count cells model is that if the cells secrete little or no factor, huge groups will form. To test the hypothesis that countin is part of such a factor, we disrupted the countin gene using homologous recombination. The countin cells did not secrete any measurable CF activity according to the bioassay, and the streams of countin cells did not break into groups, resulting in the streams coalescing into huge mounds of cells which then formed huge fruiting bodies (Brock and Gomer, 1999
). Addition of anti-countin antibodies to developing wild-type cells caused them to form large groups, again indicating that countin is used by wild-type cells for group size regulation. A secreted protein with
40% identity to countin, countin2, also regulates group size (Okuwa et al., 2001
).
To try to understand what could cause a stream of cells to break up into groups, we wrote a simple computer simulation of cells moving in a stream while secreting and sensing a diffusible factor (Roisin-Bouffay et al., 2000). This simulation indicated that if a secreted factor negatively regulates cell-cell adhesion, and/or increases random motility, the presence of too many cells in a stream results in a high level of the factor and thereby cause the stream to dissipate and subsequently break up. The subsequent lower concentrations of the factor amongst the dispersed cells will cause them to recoalesce, and the simulation indicated that they would coalesce into separate groups rather than in a continuous stream (Roisin-Bouffay et al., 2000
). During early Dictyostelium development, there are two main cell-cell adhesion molecules: gp24 and gp80 (Loomis, 1988
; Siu et al., 1987
; Siu and Kamboj, 1990
). Overexpression of gp80 caused the formation of unbroken streams and large aggregates, while blocking gp80 binding activity with monoclonal antibodies resulted in broken streams and many small aggregates (Kamboj et al., 1990
; Siu and Kamboj, 1990
). Because altering cell-cell adhesion can alter group size in Dictyostelium, we examined whether countin or smlA cells have altered cell-cell adhesion. We found that countin cells have abnormally high cell-cell adhesion, while smlA cells have low adhesion (Roisin-Bouffay et al., 2000
). Addition of highly purified preparations of CF to wild-type cells decreased cell-cell adhesion within 30 minutes. Decreasing adhesion with exogenous anti-gp24 antibodies also decreased group size. The simulations indicated that if a secreted factor regulates both adhesion and motility, the group size becomes more uniform (Tang et al., 2002
). We found that CF increases cell motility by increasing actin polymerization and the levels of the actin-crosslinking protein ABP-120, and by decreasing levels of myosin polymerization (Tang et al., 2002
). Motility and the expression of the adhesion molecules is regulated by the relayed pulses of cAMP, and we found that CF indeed regulates cAMP signal transduction (Tang et al., 2001
). These results suggested that CF regulates cell-cell adhesion and motility, and this in turn can regulate the size of a group of cells.
All of the above work was done with either countin cells or exogenous anti-countin antibodies to inhibit countin function. However, CF contains several other proteins in addition to countin. To help understand why cell-number counting in Dictyostelium is mediated by a large complex of polypeptides, we have characterized the function of CF50, another protein component of CF.
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MATERIALS AND METHODS |
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Cell culture and group number assays
Cell culture was done according to Brock and Gomer (Brock and Gomer, 1999). The disruption of strain ctnA (also known as countin) used in this study was HDB2B/4 and will be referred to as countin. The smlA disruption strain used was HDB7YA and will be referred to as smlA. Slices of agar with adhering fruiting bodies were turned sideways and photographed on Kodak Ektachrome 160T with a light blue filter using a Nikon Microphot with a 4x lens stopped down with a 1 mm pinhole in front of the lens. Synergy assays were done according to Brock et al. (Brock et al., 1996
). Production of conditioned starvation medium (CM), exposure of cells to conditioned medium, and exposure of cells to 1:500 anti-countin antibodies or 1:500 anti-CF50 antibodies was done according to Brock and Gomer (Brock and Gomer, 1999
). Group size assays were done following the method of Brock et al. (Brock et al., 1996
). To make conditioned growth medium, cells were inoculated into growth medium at 1x106 cells/ml. After 20 hours, the medium was clarified by centrifugation at 2100 g for 3 minutes, and the supernatant was further clarified by centrifugation at 12,000 g for 10 minutes. The supernatant was mixed with SDS sample buffer and heated to 100°C for 3 minutes for western blots stained with anti-CF50. For western blots stained with anti-countin antibodies, the supernatant was concentrated by a factor of 10 using an Ultrafree Biomax10 kDa cutoff spin filter (Millipore, Bedford, MA) and then treated as above. The procedure of Wood et al. (Wood et al., 1996
) was used to determine the percentages of CP2-positive and SP70-positive cells at low cell density. For each cell type, cells were grown in shaking culture, and (where indicated) recombinant CF50 was added to the cells to 0.2 µg/ml at the time indicated. The cells were then collected by centrifugation, washed in starvation buffer and starved in duplicate wells of an 8 well slide at low cell density in monolayer culture in the presence of a 1:10 dilution of Ax4 conditioned medium to supply CMF and thus allow CP2 and SP70 expression at low cell density. After 6 hours, cAMP was added to induce the expression of prestalk and prespore genes. 12 hours later, the cells were fixed and one well was stained for CP2 while the other was stained for SP70. The total number of cells per well and the number of positive cells were then counted. To examine differentiation in aggregates, cells were starved on filter pads (Brock et al., 1996
) and were dissociated after 18 hours. Approximately 2500 cells in 200 µl of PBM were placed in the well of an 8 well slide. After allowing the cells to settle for 10 minutes, the cells were fixed and stained following Wood et al. (Wood et al., 1996
).
Northern blots
RNA was isolated using a RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturers directions with the exception that 2x107 cells were used. Northern blot analysis was as described previously (Brock et al., 1996).
Preparation of recombinant CF50 and lysozyme assay
To make recombinant CF50, a PCR reaction was done using a vegetative cDNA library and the primers GGCAGCCATATGGAATGTGCCATTGATTTCTC and GGATCCTCGAGTTAAATGGATGATCCACTTCC to generate a fragment of the cf50 coding region corresponding to the entire polypeptide of the secreted protein, with a NdeI site on one end and a XhoI site on the other. To make a recombinant fragment of CF50 lacking the C-terminal serine-glycine rich region (which is very similar to a region in a 45 kDa component of CF; D. A. B., R. D. H. and R. H. G., unpublished), a PCR reaction was carried out using the first primer above and GCCGGATCCTCGAGTTAATAAAAATCTAAATCAACAC. After digestion with NdeI and XhoI, these were ligated into the corresponding sites of pET-15b (Novagen). The recombinant proteins were expressed in bacteria. The recombinant CF50 was found in inclusion bodies, so these were solubilized in 8 M urea and the recombinant CF50 was purified using a B-Per 6x His spin purification kit (Pierce, Rockford, IL) following the manufacturers directions. To refold denatured recombinant CF50, a final concentration of 100 µg/ml protein was dialyzed in 20 mM Tris-HCl, pH 7.5, 0.1 mM DTT at 4°C for 24 hours with three changes of buffer. The protein was then dialyzed in the same buffer without DTT at 4°C for 48 hours with four changes of buffer, and stored as aliquots at 80°C. The purified protein was quantitated in two ways. First, dilution curves of known amounts of bovine serum albumin and various dilutions of recombinant CF50 were electrophoresed side-by-side on SDS-polyacrylamide gels and stained with Coomassie Blue, and the lanes were scanned. Second, a Bio-Rad protein assay (Bio-Rad, Hercules, CA) was performed. The two methods gave similar results. Both constructs generated proteins that had poly-histidine tags at the N terminus, a thrombin cleavage site, followed by the first amino acid of the secreted form of CF50 (Fig. 1, first vertical arrow). The first construct generated a protein that terminated after the last amino acid of the predicted sequence. The second construct generated a protein that terminated after the last amino acid before the serine/glycine-rich motif (Fig. 1, second vertical arrow). Proteins were assayed for lysozyme activity using the method of Monchois et al. (Monchois et al., 2001) using hens egg white lysozyme (Sigma) as a standard.
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Analytical ultracentrifugation
Sedimentation equilibrium experiments were performed on a Beckman XL-A (Beckman, Palo Alto, CA) analytical ultracentrifuge with a four-position An60Ti rotor and double-sector centerpieces at 25°C. 1.0, 0.5, and 0.25 mg/ml of recombinant CF50 were examined in a six-channel centerpiece unit in which three channels on one side contained the different concentrations of protein and the three channels on the other side contained buffer. Samples were centrifuged at 10,000 rpm, 14,000 rpm, or 18,000 rpm. Analysis of data was accomplished using software provided by Beckman Instruments.
Antibody production, western blots and immunofluorescence
For antibody production, the recombinant truncated CF50 was additionally purified by SDS-polyacrylamide gel electrophoresis (Gomer, 1987). Immunization of a rabbit with the recombinant truncated CF50 and affinity purification of the antibodies was done by Bethyl Laboratories (Montgomery, TX). For western blots, 106 cells in 80 µl, or 80 µl of CM, was mixed with 20 µl of 5x Laemmli sample buffer and heated to 100°C for 3 minutes. 15 µl was electrophoresed on a 12% polyacrylamide Tris-HCl gel (Bio-Rad, Hercules, CA). Protein was transferred to Immobilon P and stained as described for the ubiquitin western blots by Lindsey et al. (Lindsey et al., 1998
) with the exception that filters were not boiled, were blocked overnight at 4°C in PBS/5% BSA/1% Tween-20/1% Nonidet P-40/0.1% SDS, and the primary antibody was used at a 1:3000 dilution. Staining of western blots with anti-countin antibodies and size-exclusion gel chromatography was performed as described in Brock and Gomer (Brock and Gomer, 1999
). For immunofluorescence, 200 µl of cells at a density of 5x105 cells/ml were placed in the well of a Lab-Tek eight-well slide (Nalge, Naperville, IL). After 30 minutes, the liquid was gently removed from the settled cells and replaced with 2% formaldehyde, 0.2% glutaraldehyde, 0.002% Triton X-100 in PBM. After 7 minutes, this was removed and the cells were washed with two changes of PBM and then incubated for 15 minutes in several changes of 4 mg/ml sodium borohydride. The cells were then stained with 10 µg/ml of affinity-purified anti-CF50 antibody (Gomer, 1987
). Cells were examined with a Nikon Microphot Fx with a 1.4 NA 60x lens or a Deltavision (Applied Precision, Issaquah, WA) deconvolution microscope with a Zeiss 1.4 NA 100x lens. Sieving gel chromatography was done as described by Brock and Gomer (Brock and Gomer, 1999
).
Cell-cell adhesion and motility
Adhesion was measured (Roisin-Bouffay et al., 2000) using cells developing on filter pads and the cells were then dissociated before doing the assay following (Tang et al., 2002
). To measure motility, cells were starved in PBM as described above, diluted to 3x105 cells/ml in PBM and 400 µl was placed in the well of a Lab-Tek 155411 8 well chambered coverglass (Nalge, Naperville, IL). After 30 minutes, 250 µl of liquid above the cells was removed from the well. Cells were videotaped for 10 minutes, 5 to 6 hours after being placed in the well, using the method of Yuen et al. (Yuen et al., 1995
) with the exception that a 20x objective was used.
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RESULTS |
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The first 24 amino acids of the predicted CF50 protein appear to be a signal sequence (typically a lysine followed by a hydrophobic region), and are missing from the purified native protein (Fig. 1). This suggests that CF50 is a secreted protein. The predicted molecular mass of the 279 amino acid protein starting from the observed N terminus is 28.2 kDa. There are two potential N-linked glycosylation sites (shaded boxes, Fig. 1) and no significant O-linked glycosylation sites. We previously observed that other proteins secreted by Dictyostelium cells are extensively glycosylated, resulting in polypeptide backbones that are 70% of the total mass of the protein (Brock and Gomer, 1999
; Jain and Gomer, 1994
). The first 200 amino acids of CF50 show a 34% identity and a 51% similarity to the Entamoeba histolytica lysozyme II precursor, and the last 70 amino acids contain 13 copies of (A/N)SGS motifs. There are no concentrations of charged amino acids, only 3 positively charged amino acids, and the predicted pI is 3.5. There are no large regions of hydrophobicity. Recombinant CF50 (which as described below has CF bioactivity) appeared to contain a mixture of
helix, ß sheet and random coil as determined by circular dichroism (data not shown), and ultracentrifugation indicated a molecular mass of 35 kDa, suggesting that the recombinant protein behaves as a monomer. Lysozyme activity assays indicated that recombinant CF50 has more than 104-fold lower specific enzymatic activity than hens egg-white lysozyme (Table 1), suggesting that despite the sequence similarity, CF50 is not a lysozyme.
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cf50 cells secrete bioactive countin activity
We previously observed that when wild-type cells were starved on filters soaked with anti-countin antibodies, the number of groups decreased and the group size increased (Brock and Gomer, 1999). To determine the effect of depleting countin and CF50 simultaneously, we starved Ax2 parental and cf50 cells on filters for 2 hours and then transferred the filters with cells to pads soaked with buffer, preimmune or immune anti-countin antibodies. There was no significant difference in the number of aggregates formed on buffer as opposed to preimmune sera. Anti-countin antibodies decreased the number of groups formed by cf50 cells, and anti-CF50 antibodies decreased the number of groups formed by wild-type cells but not cf50 cells (Table 2). The anti-CF50 antibodies also caused a slight reduction in the number of groups formed by countin cells. The data suggest that there is some residual CF activity secreted by cf50 cells, and that depleting extracellular countin can decrease this activity. The data also indicate that anti-CF50 antibodies are able to deplete CF activity from the extracellular medium, and that countin cells secrete a small amount of CF activity that can be neutralized with anti-CF50 antibodies.
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Like countin cells, cf50 cells have high cell-cell adhesion and low motility
Computer simulations predicted that a higher cell-cell adhesion and/or a lower random cell motility would keep a stream intact, and thus by preventing stream breakup lead to larger groups (Roisin-Bouffay et al., 2000). We previously observed that countin cells have a higher cell-cell adhesion than their parental cells. Compared to wild-type parental, cf50 cells had 5.9±1.5% higher adhesion at 2 hours of development, 3.0±1.3% higher at 4 hours, and 2.2±0.9% higher at 6 hours (means ± s.e.m. from 5 separate experiments). These experiments were done on cells that were forming streams on filter pads. The streams were morphologically similar up to about 8 hours of development, and had not begun to coalesce. Thus whereas during later development the cf50 cells form huge groups and cells in the interior might be starved for oxygen, in these assays the cells were in structures of a similar size. In addition to having abnormally high cell-cell adhesion, countin cells have an abnormally low cell motility during the time wild-type streams are breaking up (Tang et al., 2002
). cf50 cells also have significantly decreased cell motility at 6 hours after starvation (Fig. 9). These assays were done with cells developing in submerged monolayer culture, so, as above, these differences are not due to factors such as a lack of oxygen. Together, the data indicate that disruption of cf50 has qualitatively the same effect on cell-cell adhesion and cell motility as disruption of countin.
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DISCUSSION |
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Native CF50 is a 50 kDa molecule, whereas the polypeptide backbone is only 28 kDa. Since many secreted proteins in Dictyostelium are glycosylated, one possibility is that the 22 kDa of non-polypeptide mass is post-translational glycosylation. Our observation that bacterially synthesized CF50 polypeptide backbone has CF activity when added to wild-type or cf50 cells suggests that the functional domain of CF50 is in this backbone. countin cells did not exhibit a strong response to the recombinant CF50, possibly because they already secrete large amounts of CF50 (Fig. 2C). For a different secreted polypeptide factor, CMF, we found that the presence of post-translational glycosylation appears to protect the protein from proteases (Jain and Gomer, 1994; Yuen et al., 1991
). Thus a possible explanation for the existence of the CF50 glycosylation is that it protects CF50 from degradation. We previously observed that highly purified preparations of CF could cause a 3-fold increase in group number, and that a 1.5-fold increase could occur at concentration as low as 0.3 µg/ml (Brock and Gomer, 1999
). The recombinant CF50 occasionally will cause a 2-fold increase in group number, but a 1.5-fold increase tends to be the norm and the lowest concentration this occurs is at
0.1 µg/ml; Fig. 5 and data not shown). Assuming that the CF50 is roughly one-fifth of the CF, the specific activity of CF50 is roughly that of CF, although recombinant CF50 does not seem to increase group number to the extent that CF can.
Both countin and cf50 cells produce conditioned medium that causes a slight increase in group number compared to buffer alone, suggesting that there is some residual CF activity secreted in cells lacking just countin or just CF50. The antibody depletion experiments indicated that cf50 cells, which form large groups, will form even larger groups when countin is depleted from the extracellular environment with anti-countin antibodies, and countin cells similarly form slightly larger groups when CF50 is depleted with antibodies (Table 2). Thus both the CF production assays and antibody depletion experiments suggest that neither CF50 nor countin is the sole effector molecule in the CF complex, but that both molecules can separately affect group size.
During early development, countin cells have a considerably higher cell-cell adhesion than parental cells. We found that cf50 cells have only a slightly higher adhesion than parental cells. This higher adhesion is probably not sufficient to strongly affect group size. However, the cf50 cells have a significantly lower motility than parental cells, and most importantly far fewer highly motile cells than wild type (Fig. 9). Our computer simulations suggest that streams break when strongly motile cells break adhesive bonds and tear away from other cells (Roisin-Bouffay et al., 2000; Tang et al., 2002
). The highly motile cells thus have the greatest influence on stream breaking, and we hypothesize that the greatly reduced numbers of these cells seen in cf50 is the reason cf50 cells form streams that rarely break and thus form large groups.
Vegetative countin cells have high cell-cell adhesion and high levels of the adhesion molecule gp24 compared to wild-type cells, while vegetative smlA cells have lower levels (Roisin-Bouffay et al., 2000) (Roisin-Bouffay and Gomer, unpublished). Vegetative cells contain smlA and countin proteins (Brock et al., 1996
; Brock and Gomer, 1999
), and we found here that vegetative cells also contain CF50. If we assume that vegetative cells can respond to CF, our observation that countin and CF50 are secreted by vegetative cells suggests that the differences in gp24 levels and cell-cell adhesion in vegetative smlA, wild-type and countin cells could be due to low CF activity being secreted by vegetative countin cells and high levels of CF being secreted by smlA cells. This could then result in different levels of gp24 and different levels of cell-cell adhesion in vegetative wild-type cells compared to smlA or countin cells.
Wild-type cells all appear to have the same amount and distribution of CF50 (Fig. 7), so the altered cell-type differentiation of cf50 cells (Tables 3, 4, and 5) does not appear to be due to CF50 levels or distribution specifying a cell fate. This then suggests that CF50 somehow affects a process that is however heterogeneous in the cells. Although recombinant CF50 can rescue the abnormal initial cell-type differentiation when added to vegetative cf50 cells that are subsequently starved at low cell density, anti-CF50 antibodies added during starvation can block this rescue (Table 4). In addition, when the CF50-treated cells are starved at high cell density, there is very little change in the initial cell-type differentiation (Table 5). Together, the data suggest that the recombinant CF50 added to growing cf50 cells affects initial differentiation at a step after growth. One possibility is that the cf50 cells are able to sequester the added CF50 and then release it during development, and that this released CF50 can rescue the altered initial cell-type differentiation of cf50 cells when they are starved at low density but is degraded when the cells are starved at high cell density. Recombinant CF50 affects group size when added to vegetative cells that are then washed and starved at high cell density. Under these same conditions there is little effect on initial cell-type choice. A possible explanation for this is that during growth CF50 affects the expression of genes involved in adhesion and/or motility, and the altered levels of the associated proteins persist into development and affect stream breakup.
It is unclear why recombinant CF50 affects group size in Ax4 cells, which secrete CF50 as part of the CF complex. One possibility is that there are limiting amounts of CF50 in CM, and the recombinant CF50 allows CF complexes to form and or become active. However, western blots of gel filtration column fractions of whole conditioned medium from wild-type cells indicate that all of the detectable countin and CF50 are both present in a roughly 450 kDa complex, suggesting that there are no incomplete complexes. In addition, the removal of CF50 causes much of the countin present in CM to elute at a lower molecular weight (Fig. 8B). However, we cannot exclude the possibility that some fraction of countin was present in 450 kDa complexes that lack CF50, and that exogenous CF50 increased the activity of this subset of complexes. Another possibility is that the exogenous CF50 (and thus the CF50 in the CF complex) binds to and activates a cell surface receptor.
There are several examples of signals that are present in complexes. One example is a ternary complex of Twisted gastrulation (TSG)/Short gastrulation (SOG or chordin)/Bone morphogenetic protein (BMP) (Chang et al., 2001; Ross et al., 2001
; Scott et al., 2001
). TSG and SOG act synergistically to repress signaling by BMP and TSG appears to enhance a proteolytic cleavage of SOG. Insulin-like growth factors, which can regulate the size of developing tissues, are found in ternary complexes with two specific binding proteins (Boisclair et al., 2001
). Another example is TGFß, which is stored in platelets and secreted as a complex with TGFß masking protein, a 440 kDa complex of polypeptides. TGFß forms a homodimer, and the masking protein contains 4 to 6 subunits (Nakamura et al., 1986
; Okada et al., 1989
). For all three of these examples, one protein in the complex can act as a signal, and other proteins regulate its activity. Another signal that is normally in a complex is thrombospondin, a trimeric
450 kDa secreted extracellular matrix protein with binding sites for a variety of factors and receptors (Bornstein et al., 2000
; Chen et al., 2000
). Like CF, thrombospondin inhibits cell adhesion and increases cell motility, allowing cells to move and thus reshape structures (Mansfield et al., 1990
; Murphy-Ullrich, 2001
). CF appears to be somewhat different from these examples of multi-subunit factors insofar as having subunits that have similar as well as distinct effects on cells. This then suggests the possibility that for unknown reasons there may be different receptors for different components of the cell-number counting factor.
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ACKNOWLEDGMENTS |
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