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A role for syndecan-1 in coupling fascin spike formation by thrombospondin-1.

Adams JC, Kureishy N, Taylor AL - J. Cell Biol. (2001)

Bottom Line: The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation.Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1.These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom. dmcbjca@ucl.ac.uk

ABSTRACT
An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.

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Effects of syndecan expression on aspects of cell motile behavior. Apical projections and lateral protrusive ruffling activity of cells adherent on 50 nM TSP-1 were scored from phase–contrast time-lapse videos that tracked vector or syndecan transfectant cells from 45 min to 1.5 h of adhesion. Locomotion was scored as the displacement of cell centroids between 45 min and 2 h of adhesion. Bars show mean ± SEM for data from five experiments. Shade code same as in Fig. 7.
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Figure 10: Effects of syndecan expression on aspects of cell motile behavior. Apical projections and lateral protrusive ruffling activity of cells adherent on 50 nM TSP-1 were scored from phase–contrast time-lapse videos that tracked vector or syndecan transfectant cells from 45 min to 1.5 h of adhesion. Locomotion was scored as the displacement of cell centroids between 45 min and 2 h of adhesion. Bars show mean ± SEM for data from five experiments. Shade code same as in Fig. 7.

Mentions: In addition to their role in cell adhesion to TSP-1, fascin spikes support cell motility on TSP-1 matrix (Adams 1997a,Adams 1997b; Adams and Schwartz 2000). We undertook time-lapse experiments to examine the motile behavior of the syndecan-1–transfected COS-7 cells on TSP-1 or on syndecan-1 antibody-coated surfaces. Cells were scored for two aspects of motility: protrusive lateral ruffling activity and locomotion over the substratum. When attached to TSP-1, few of the vector transfectant cells showed extension of spikes in contact with the substratum, there was little ruffling activity at lateral cell margins, and all cells were nonlocomotary (Fig. 10). In marked contrast, the syndecan-1 transfectant cells showed greatly increased ruffling at their lateral margins (Fig. 10; significant at P > 0.001). These lateral ruffles appeared as dynamic phase dark regions that protruded rapidly along the cell margin or moved centripetally across the spread lamellae. These were also apparent in syndecan-1 transfectants adherent on syndecan-1 antibody (data not shown). Cells expressing Syn-1/TGM also showed highly significant increases in lateral ruffling (Fig. 10). In contrast, cells transfected with the syn-1/ΔVC2 construct did not show lateral ruffles and thus appeared similar to vector transfectants (data not shown). To examine the specificity of the ruffling response, we compared the motility of cells transfected with syndecan-2. These cells showed no lateral ruffling. Lateral ruffling thus specifically correlated with the formation of fascin spikes (Fig. 7 B and Fig. 10). The alterations in lateral ruffling occurred in the absence of any change in extension of apical projections, and thus related specifically to adhesion-dependent motility and not to a general change in membrane dynamics (Fig. 10). Interestingly, despite the large increases in cell spreading and lateral ruffling activity, syndecan-1 transfectants did not translocate over the substratum. For adhesion to TSP-1, this result was obtained using coating concentrations of 25 or 50 nM. For adhesion to the syndecan-1 antibody, this result was confirmed at a range of antibody-coating concentrations from 5 to 50 μg/ml. For all concentrations that supported cell spreading, no stimulation of cell locomotion was observed (data not shown). Thus, under these experimental conditions, the most dramatic effect of syndecan-1 ligation on motile behavior is the stimulation of active ruffling protrusions at lateral cell margins (Fig. 10).


A role for syndecan-1 in coupling fascin spike formation by thrombospondin-1.

Adams JC, Kureishy N, Taylor AL - J. Cell Biol. (2001)

Effects of syndecan expression on aspects of cell motile behavior. Apical projections and lateral protrusive ruffling activity of cells adherent on 50 nM TSP-1 were scored from phase–contrast time-lapse videos that tracked vector or syndecan transfectant cells from 45 min to 1.5 h of adhesion. Locomotion was scored as the displacement of cell centroids between 45 min and 2 h of adhesion. Bars show mean ± SEM for data from five experiments. Shade code same as in Fig. 7.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199199&req=5

Figure 10: Effects of syndecan expression on aspects of cell motile behavior. Apical projections and lateral protrusive ruffling activity of cells adherent on 50 nM TSP-1 were scored from phase–contrast time-lapse videos that tracked vector or syndecan transfectant cells from 45 min to 1.5 h of adhesion. Locomotion was scored as the displacement of cell centroids between 45 min and 2 h of adhesion. Bars show mean ± SEM for data from five experiments. Shade code same as in Fig. 7.
Mentions: In addition to their role in cell adhesion to TSP-1, fascin spikes support cell motility on TSP-1 matrix (Adams 1997a,Adams 1997b; Adams and Schwartz 2000). We undertook time-lapse experiments to examine the motile behavior of the syndecan-1–transfected COS-7 cells on TSP-1 or on syndecan-1 antibody-coated surfaces. Cells were scored for two aspects of motility: protrusive lateral ruffling activity and locomotion over the substratum. When attached to TSP-1, few of the vector transfectant cells showed extension of spikes in contact with the substratum, there was little ruffling activity at lateral cell margins, and all cells were nonlocomotary (Fig. 10). In marked contrast, the syndecan-1 transfectant cells showed greatly increased ruffling at their lateral margins (Fig. 10; significant at P > 0.001). These lateral ruffles appeared as dynamic phase dark regions that protruded rapidly along the cell margin or moved centripetally across the spread lamellae. These were also apparent in syndecan-1 transfectants adherent on syndecan-1 antibody (data not shown). Cells expressing Syn-1/TGM also showed highly significant increases in lateral ruffling (Fig. 10). In contrast, cells transfected with the syn-1/ΔVC2 construct did not show lateral ruffles and thus appeared similar to vector transfectants (data not shown). To examine the specificity of the ruffling response, we compared the motility of cells transfected with syndecan-2. These cells showed no lateral ruffling. Lateral ruffling thus specifically correlated with the formation of fascin spikes (Fig. 7 B and Fig. 10). The alterations in lateral ruffling occurred in the absence of any change in extension of apical projections, and thus related specifically to adhesion-dependent motility and not to a general change in membrane dynamics (Fig. 10). Interestingly, despite the large increases in cell spreading and lateral ruffling activity, syndecan-1 transfectants did not translocate over the substratum. For adhesion to TSP-1, this result was obtained using coating concentrations of 25 or 50 nM. For adhesion to the syndecan-1 antibody, this result was confirmed at a range of antibody-coating concentrations from 5 to 50 μg/ml. For all concentrations that supported cell spreading, no stimulation of cell locomotion was observed (data not shown). Thus, under these experimental conditions, the most dramatic effect of syndecan-1 ligation on motile behavior is the stimulation of active ruffling protrusions at lateral cell margins (Fig. 10).

Bottom Line: The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation.Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1.These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom. dmcbjca@ucl.ac.uk

ABSTRACT
An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.

Show MeSH
Related in: MedlinePlus