Abstract
The laminin-based nonapeptide Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR) and pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR) have been previously demonstrated to support the attachment of several cell types and to competitively bind to the 67-kDa high affinity laminin receptor. Cell attachment, but not spreading, on substrates containing adsorbed CDPGYIGSR or YIGSR was observed. In this report we describe YIGSR-mediated attachment and spreading of a wide variety of cell types. GYIGSRY promoted cell spreading and stress fiber formation when it was covalently immobilized through the amino-terminal Gly residue, used as a spacer arm. Spreading was not observed when adsorbed YIGSR peptide was used. Functionally blocking antiserum directed against the 67-kDa and related laminin-binding proteins blocked human foreskin fibroblast (HFF) spreading, but not attachment, on covalently grafted GYIGSRY substrates. However, functionally blocking antisera directed against the vitronectin receptor, integrin alpha v beta 3, and the fibronectin receptor, integrin alpha 5 beta 1, did not affect HFF spreading on these substrates. When HFFs spread on these substrates, the 67-kDa laminin receptor co-localized with the cytoplasmic proteins alpha-actinin and vinculin into discrete structures. These results suggest that the adhesion ligand YIGSR is solely sufficient for cell spreading when it is conformationally constrained by covalent attachment to a solid substrate, at least when attached via its amino terminus. Furthermore, the role of the 67-kDa laminin receptor in recognition of this ligand and mediating cell attachment is confirmed in this study. This report also provides the first evidence for direct or indirect association of this receptor with vinculin and alpha-actinin when YIGSR-mediated cell spreading occurs.
Highlights
Immobilized Laminin Peptide Tyr-Ile-Gly-Ser-Arg (YIGSR) Supports Cell Spreading andCo-localization of the 67Kilodalton Laminin Receptor with a-Actiniannd Vinculin*
The extentof cell spreading on GYIGSRY-grafted substratesfor the remainderof the cell types tested was generally comparable to that on fibronectin- and laminin-coated substrates (Table11)
Actin microfilament bundle formation was observed in the other cells which spread on grafted GYIGSRY
Summary
Cell Spreadingand Cytoskeletal Organization-All cell types examined, exceptfor rat calvarium osteoblastsw,ere observed tospreadonGYIGSRY-graftedsubstrates(Table. The extent of cell spreading on GYIGSRY-grafted substrateswas quite low for rabbit corneal epithelial cells and the MG 63 humanosteosarcoma cell line. Cell spreading was markedly increased for these cell types on the lamininand fibronectin-coated substrates. The extentof cell spreading on GYIGSRY-grafted substratesfor the remainderof the cell types tested was generally comparable to that on fibronectin- and laminin-coated substrates (Table). Most noteworthy of the cell spreadingresults was the lack of cell spreadingonadsorbed GYIGSRY. Cell spreading occurred onlywhenGYIGSRY was covalently boundto glass substrates.Rhodamine-phalloidinstaining revealedextensive stressfiberformationwithinspreadHFFs (Fig. lA) and HUVECs (Fig. 1B).Characteristically dense peripheral staining of actin microfilament bundles wasobservedin rabbit corneal epithelial cells (Fig. 1C). Actin microfilament bundle formation was observed in the other cells which spread on grafted GYIGSRY (data not shown)
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