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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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GAG modifications of syndecan-1 and regions of the cytoplasmic domain are required for fascin spike formation on TSP-1. COS-7 cells were transfected with the indicated syndecan-1 mutants and EGFP-fascin expression plasmids, and 48 h later EDTA-released cells were plated on 50 nM TSP-1 in serum-free medium for 1 h and then fixed and stained for F-actin. Results shown are representative of three experiments, and ≥100 transfected cells were scored per experiment. Bar, 16 μm.
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Figure 6: GAG modifications of syndecan-1 and regions of the cytoplasmic domain are required for fascin spike formation on TSP-1. COS-7 cells were transfected with the indicated syndecan-1 mutants and EGFP-fascin expression plasmids, and 48 h later EDTA-released cells were plated on 50 nM TSP-1 in serum-free medium for 1 h and then fixed and stained for F-actin. Results shown are representative of three experiments, and ≥100 transfected cells were scored per experiment. Bar, 16 μm.

Mentions: The extracellular domain of syndecan-1 proteoglycan contains three conserved sites for GAG substitution near the NH2 terminus. These are sites of addition for HS-GAGs in many cell types but can also be substituted by CS-GAGs (Kokenyesi and Bernfield 1994). HS-GAG chains have well-established functional roles in the activities of syndecans, particularly in binding growth factors such as FGF (for reviews see Rapraeger and Ott 1998; Bernfield et al. 1999). To determine whether GAGs also contribute to cytoskeletal coupling by syndecan-1, COS-7 cells were transiently cotransfected with EGFP-fascin and a syndecan-1 expression construct in which the three sites of GAG addition, serines 37, 45, and 47, had been mutated to alanines, syn-1/TGM (Langford et al. 1998). Strikingly, these cells spread poorly on TSP-1 compared with the wild-type syndecan-1 transfectants, and lamellipodia were not observed. Instead, the cells formed actin- and fascin-containing projections that appeared long in comparison to the wild-type syndecan-1 transfectants (Fig. 6). These parameters were quantitated by image analysis measurements. The mean cell area of syn-1/TGM transfectants was not significantly increased compared with vector control transfectants. However, the number of spikes per cell and their length were strongly increased (significant at P = 0.001; Fig. 7A and Fig. B). Compared with cells expressing wild-type syndecan-1, the number of spikes per cell was somewhat increased (significant at P = 0.04), and the mean length of spikes was increased from 8.5 to 13.5 μm (significant at P = 0.01; Fig. 7 B).


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

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

GAG modifications of syndecan-1 and regions of the cytoplasmic domain are required for fascin spike formation on TSP-1. COS-7 cells were transfected with the indicated syndecan-1 mutants and EGFP-fascin expression plasmids, and 48 h later EDTA-released cells were plated on 50 nM TSP-1 in serum-free medium for 1 h and then fixed and stained for F-actin. Results shown are representative of three experiments, and ≥100 transfected cells were scored per experiment. Bar, 16 μm.
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Related In: Results  -  Collection

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Figure 6: GAG modifications of syndecan-1 and regions of the cytoplasmic domain are required for fascin spike formation on TSP-1. COS-7 cells were transfected with the indicated syndecan-1 mutants and EGFP-fascin expression plasmids, and 48 h later EDTA-released cells were plated on 50 nM TSP-1 in serum-free medium for 1 h and then fixed and stained for F-actin. Results shown are representative of three experiments, and ≥100 transfected cells were scored per experiment. Bar, 16 μm.
Mentions: The extracellular domain of syndecan-1 proteoglycan contains three conserved sites for GAG substitution near the NH2 terminus. These are sites of addition for HS-GAGs in many cell types but can also be substituted by CS-GAGs (Kokenyesi and Bernfield 1994). HS-GAG chains have well-established functional roles in the activities of syndecans, particularly in binding growth factors such as FGF (for reviews see Rapraeger and Ott 1998; Bernfield et al. 1999). To determine whether GAGs also contribute to cytoskeletal coupling by syndecan-1, COS-7 cells were transiently cotransfected with EGFP-fascin and a syndecan-1 expression construct in which the three sites of GAG addition, serines 37, 45, and 47, had been mutated to alanines, syn-1/TGM (Langford et al. 1998). Strikingly, these cells spread poorly on TSP-1 compared with the wild-type syndecan-1 transfectants, and lamellipodia were not observed. Instead, the cells formed actin- and fascin-containing projections that appeared long in comparison to the wild-type syndecan-1 transfectants (Fig. 6). These parameters were quantitated by image analysis measurements. The mean cell area of syn-1/TGM transfectants was not significantly increased compared with vector control transfectants. However, the number of spikes per cell and their length were strongly increased (significant at P = 0.001; Fig. 7A and Fig. B). Compared with cells expressing wild-type syndecan-1, the number of spikes per cell was somewhat increased (significant at P = 0.04), and the mean length of spikes was increased from 8.5 to 13.5 μm (significant at P = 0.01; Fig. 7 B).

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