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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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Expression of TSP-1–binding receptors by C2C12 cells. (A) FACS® profiles. C2C12 cells were disaggregated with EDTA or with trypsin and stained with antibodies to the indicated adhesion receptors or nonimmune (NI) rat immunoglobulin. Similar profiles were obtained for EDTA (E) or trypsin-disaggregated (T) cells with the CD47, β1, and β3 antibodies (both profiles shown for β1 integrin only), whereas cells trypsinized for 2 min showed more heterogeneous syndecan-1 staining. (B) Syndecan-1 is expressed on C2C12 cells as a mixed proteoglycan. Proteoglycan extracts were prepared from C2C12 cells, resolved on gradient polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and probed with antibody 281-2 to mouse syndecan-1. Lane 1, undigested extract; lane 2, + chondroitinase ABC digestion; lane 3, + heparitinase II digestion; lane 4, + digestion with both enzymes. Markers are indicated in kD. Results are representative of three experiments.
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Figure 1: Expression of TSP-1–binding receptors by C2C12 cells. (A) FACS® profiles. C2C12 cells were disaggregated with EDTA or with trypsin and stained with antibodies to the indicated adhesion receptors or nonimmune (NI) rat immunoglobulin. Similar profiles were obtained for EDTA (E) or trypsin-disaggregated (T) cells with the CD47, β1, and β3 antibodies (both profiles shown for β1 integrin only), whereas cells trypsinized for 2 min showed more heterogeneous syndecan-1 staining. (B) Syndecan-1 is expressed on C2C12 cells as a mixed proteoglycan. Proteoglycan extracts were prepared from C2C12 cells, resolved on gradient polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and probed with antibody 281-2 to mouse syndecan-1. Lane 1, undigested extract; lane 2, + chondroitinase ABC digestion; lane 3, + heparitinase II digestion; lane 4, + digestion with both enzymes. Markers are indicated in kD. Results are representative of three experiments.

Mentions: C2C12 skeletal myoblasts attach to TSP-1 through predominant interactions with the type 1 repeats and COOH-terminal domain (Adams and Lawler 1994). With the aim of identifying the adhesive receptors which couple cell attachment to fascin spike organization, we first characterized the expression by C2C12 cells of receptors that are involved in cell attachment to TSP-1 in multiple cell types. FACS® analysis was carried out on live cell suspensions which had been disaggregated either by treatment with 10 mM EDTA or by trypsinization. EDTA-released C2C12 cells expressed β1 and β3 integrin subunits and also CD47 and syndecan-1 but did not express detectable CD36 (Fig. 1 A; data not shown). Direct analysis of integrin heterodimers by immunoprecipitation of surface-labeled cells established expression of α2β1, α3β1, α4β1, α5β1, α7β1, and αvβ1 but not α1β1 integrins. The cells also express the αvβ3 and αvβ5 heterodimers (Adams et al. 1998 and references therein; Adams, J.C., unpublished data). The expression profiles for CD47 and the integrin subunits were very similar in trypsinized or EDTA-treated cells (shown for β1 staining only; Fig. 1 A). However, after trypsin treatment for 2 min at 37°C expression of syndecan-1 was reduced and more heterogeneous (Fig. 1 A, panel Syn-1[T]). As expected, because of the known protease sensitivity of the syndecan-1 extracellular domain, trypsinization for 5 min at 37°C resulted in a complete loss of cell surface syndecan-1 (data not shown; Fitzgerald et al. 2000).


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

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

Expression of TSP-1–binding receptors by C2C12 cells. (A) FACS® profiles. C2C12 cells were disaggregated with EDTA or with trypsin and stained with antibodies to the indicated adhesion receptors or nonimmune (NI) rat immunoglobulin. Similar profiles were obtained for EDTA (E) or trypsin-disaggregated (T) cells with the CD47, β1, and β3 antibodies (both profiles shown for β1 integrin only), whereas cells trypsinized for 2 min showed more heterogeneous syndecan-1 staining. (B) Syndecan-1 is expressed on C2C12 cells as a mixed proteoglycan. Proteoglycan extracts were prepared from C2C12 cells, resolved on gradient polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and probed with antibody 281-2 to mouse syndecan-1. Lane 1, undigested extract; lane 2, + chondroitinase ABC digestion; lane 3, + heparitinase II digestion; lane 4, + digestion with both enzymes. Markers are indicated in kD. Results are representative of three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199199&req=5

Figure 1: Expression of TSP-1–binding receptors by C2C12 cells. (A) FACS® profiles. C2C12 cells were disaggregated with EDTA or with trypsin and stained with antibodies to the indicated adhesion receptors or nonimmune (NI) rat immunoglobulin. Similar profiles were obtained for EDTA (E) or trypsin-disaggregated (T) cells with the CD47, β1, and β3 antibodies (both profiles shown for β1 integrin only), whereas cells trypsinized for 2 min showed more heterogeneous syndecan-1 staining. (B) Syndecan-1 is expressed on C2C12 cells as a mixed proteoglycan. Proteoglycan extracts were prepared from C2C12 cells, resolved on gradient polyacrylamide gels under reducing conditions, transferred to nitrocellulose, and probed with antibody 281-2 to mouse syndecan-1. Lane 1, undigested extract; lane 2, + chondroitinase ABC digestion; lane 3, + heparitinase II digestion; lane 4, + digestion with both enzymes. Markers are indicated in kD. Results are representative of three experiments.
Mentions: C2C12 skeletal myoblasts attach to TSP-1 through predominant interactions with the type 1 repeats and COOH-terminal domain (Adams and Lawler 1994). With the aim of identifying the adhesive receptors which couple cell attachment to fascin spike organization, we first characterized the expression by C2C12 cells of receptors that are involved in cell attachment to TSP-1 in multiple cell types. FACS® analysis was carried out on live cell suspensions which had been disaggregated either by treatment with 10 mM EDTA or by trypsinization. EDTA-released C2C12 cells expressed β1 and β3 integrin subunits and also CD47 and syndecan-1 but did not express detectable CD36 (Fig. 1 A; data not shown). Direct analysis of integrin heterodimers by immunoprecipitation of surface-labeled cells established expression of α2β1, α3β1, α4β1, α5β1, α7β1, and αvβ1 but not α1β1 integrins. The cells also express the αvβ3 and αvβ5 heterodimers (Adams et al. 1998 and references therein; Adams, J.C., unpublished data). The expression profiles for CD47 and the integrin subunits were very similar in trypsinized or EDTA-treated cells (shown for β1 staining only; Fig. 1 A). However, after trypsin treatment for 2 min at 37°C expression of syndecan-1 was reduced and more heterogeneous (Fig. 1 A, panel Syn-1[T]). As expected, because of the known protease sensitivity of the syndecan-1 extracellular domain, trypsinization for 5 min at 37°C resulted in a complete loss of cell surface syndecan-1 (data not shown; Fitzgerald et al. 2000).

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