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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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The effects of GAG removal from syndecan-1 are specific to TSP-1 adhesion. (A) COS-7 cells transfected for 48 h with the indicated syndecan constructs and EGFP-fascin were EDTA-released for 1-h adhesion assays on antibody to mouse syndecan-1 or 50 nM fibronectin. Actin organization was compared with EGFP-fascin distribution in the transfected cells. Results shown are representative of three experiments, and ≥50 transfected cells were scored per experiment. (B) Relative expression levels of syndecans used in this study. Top, in vitro translation of 1 μg of each syndecan construct detected by [35S]methionine incorporation; bottom, immunoblot of double heparitinase and chondroitinase–digested syndecan-1 proteins after expression and extraction from COS-7 cells detected by 281-2 antibody. Molecular weight markers are given in kD. Bar, 10 μm.
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Figure 8: The effects of GAG removal from syndecan-1 are specific to TSP-1 adhesion. (A) COS-7 cells transfected for 48 h with the indicated syndecan constructs and EGFP-fascin were EDTA-released for 1-h adhesion assays on antibody to mouse syndecan-1 or 50 nM fibronectin. Actin organization was compared with EGFP-fascin distribution in the transfected cells. Results shown are representative of three experiments, and ≥50 transfected cells were scored per experiment. (B) Relative expression levels of syndecans used in this study. Top, in vitro translation of 1 μg of each syndecan construct detected by [35S]methionine incorporation; bottom, immunoblot of double heparitinase and chondroitinase–digested syndecan-1 proteins after expression and extraction from COS-7 cells detected by 281-2 antibody. Molecular weight markers are given in kD. Bar, 10 μm.

Mentions: To determine whether these effects were specific to TSP-1 ligation of syndecan-1, we examined the responses of the transfectants to plating on syndecan antibody or on fibronectin. On the syndecan-1 antibody, the syn-1/TGM transfectants spread and organized fascin–actin bundles, whereas the syn-1/ΔVC2 transfectants remained round as they did on TSP-1 (Fig. 8 A). Thus, even upon direct, monospecific ligation of the core protein the V and C2 cytoplasmic regions are needed to transduce cytoskeletal organization. To further confirm that the effects of syndecan-1 expression cells resulted from specific ligation of syndecan-1 and not as an immediate consequence of syndecan overexpression, we examined cell behavior on fibronectin. No alterations in cell spreading or fascin distribution were apparent in cells transfected with syn-1/TGM, syn-1/ΔVC2, or syndecan-2 (Fig. 8 A). These cDNAs used were all translatable with equivalent efficiencies (Fig. 8 B), and both Syn1/TGM and Syn1/ΔVC2 proteins were expressed at comparable levels to the wild-type syndecan-1 as determined by FACS® analysis or immunoblot (Fig. 8 B; data not shown). Thus, the differing effects of the various syndecan-1 proteins on fascin organization most likely result from ligation of syndecan-1 by appropriate specific ligands.


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

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

The effects of GAG removal from syndecan-1 are specific to TSP-1 adhesion. (A) COS-7 cells transfected for 48 h with the indicated syndecan constructs and EGFP-fascin were EDTA-released for 1-h adhesion assays on antibody to mouse syndecan-1 or 50 nM fibronectin. Actin organization was compared with EGFP-fascin distribution in the transfected cells. Results shown are representative of three experiments, and ≥50 transfected cells were scored per experiment. (B) Relative expression levels of syndecans used in this study. Top, in vitro translation of 1 μg of each syndecan construct detected by [35S]methionine incorporation; bottom, immunoblot of double heparitinase and chondroitinase–digested syndecan-1 proteins after expression and extraction from COS-7 cells detected by 281-2 antibody. Molecular weight markers are given in kD. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: The effects of GAG removal from syndecan-1 are specific to TSP-1 adhesion. (A) COS-7 cells transfected for 48 h with the indicated syndecan constructs and EGFP-fascin were EDTA-released for 1-h adhesion assays on antibody to mouse syndecan-1 or 50 nM fibronectin. Actin organization was compared with EGFP-fascin distribution in the transfected cells. Results shown are representative of three experiments, and ≥50 transfected cells were scored per experiment. (B) Relative expression levels of syndecans used in this study. Top, in vitro translation of 1 μg of each syndecan construct detected by [35S]methionine incorporation; bottom, immunoblot of double heparitinase and chondroitinase–digested syndecan-1 proteins after expression and extraction from COS-7 cells detected by 281-2 antibody. Molecular weight markers are given in kD. Bar, 10 μm.
Mentions: To determine whether these effects were specific to TSP-1 ligation of syndecan-1, we examined the responses of the transfectants to plating on syndecan antibody or on fibronectin. On the syndecan-1 antibody, the syn-1/TGM transfectants spread and organized fascin–actin bundles, whereas the syn-1/ΔVC2 transfectants remained round as they did on TSP-1 (Fig. 8 A). Thus, even upon direct, monospecific ligation of the core protein the V and C2 cytoplasmic regions are needed to transduce cytoskeletal organization. To further confirm that the effects of syndecan-1 expression cells resulted from specific ligation of syndecan-1 and not as an immediate consequence of syndecan overexpression, we examined cell behavior on fibronectin. No alterations in cell spreading or fascin distribution were apparent in cells transfected with syn-1/TGM, syn-1/ΔVC2, or syndecan-2 (Fig. 8 A). These cDNAs used were all translatable with equivalent efficiencies (Fig. 8 B), and both Syn1/TGM and Syn1/ΔVC2 proteins were expressed at comparable levels to the wild-type syndecan-1 as determined by FACS® analysis or immunoblot (Fig. 8 B; data not shown). Thus, the differing effects of the various syndecan-1 proteins on fascin organization most likely result from ligation of syndecan-1 by appropriate specific ligands.

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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Related in: MedlinePlus