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. 1991 Sep 1;114(5):1089–1100. doi: 10.1083/jcb.114.5.1089

An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3- mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation

PMCID: PMC2289117  PMID: 1714913

Abstract

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well- defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface- bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.

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Selected References

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  1. Brandley B. K., Schnaar R. L. Covalent attachment of an Arg-Gly-Asp sequence peptide to derivatizable polyacrylamide surfaces: support of fibroblast adhesion and long-term growth. Anal Biochem. 1988 Jul;172(1):270–278. doi: 10.1016/0003-2697(88)90442-3. [DOI] [PubMed] [Google Scholar]
  2. Brandley B. K., Schnaar R. L. Tumor cell haptotaxis on covalently immobilized linear and exponential gradients of a cell adhesion peptide. Dev Biol. 1989 Sep;135(1):74–86. doi: 10.1016/0012-1606(89)90159-0. [DOI] [PubMed] [Google Scholar]
  3. Buck C. A., Horwitz A. F. Cell surface receptors for extracellular matrix molecules. Annu Rev Cell Biol. 1987;3:179–205. doi: 10.1146/annurev.cb.03.110187.001143. [DOI] [PubMed] [Google Scholar]
  4. CURTIS A. S. THE MECHANISM OF ADHESION OF CELLS TO GLASS. A STUDY BY INTERFERENCE REFLECTION MICROSCOPY. J Cell Biol. 1964 Feb;20:199–215. doi: 10.1083/jcb.20.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Danilov Y. N., Juliano R. L. (Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion. Exp Cell Res. 1989 May;182(1):186–196. doi: 10.1016/0014-4827(89)90290-5. [DOI] [PubMed] [Google Scholar]
  6. Dejana E., Colella S., Conforti G., Abbadini M., Gaboli M., Marchisio P. C. Fibronectin and vitronectin regulate the organization of their respective Arg-Gly-Asp adhesion receptors in cultured human endothelial cells. J Cell Biol. 1988 Sep;107(3):1215–1223. doi: 10.1083/jcb.107.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dejana E., Lampugnani M. G., Giorgi M., Gaboli M., Federici A. B., Ruggeri Z. M., Marchisio P. C. Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly-Asp-dependent mechanism. J Cell Biol. 1989 Jul;109(1):367–375. doi: 10.1083/jcb.109.1.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fath K. R., Edgell C. J., Burridge K. The distribution of distinct integrins in focal contacts is determined by the substratum composition. J Cell Sci. 1989 Jan;92(Pt 1):67–75. doi: 10.1242/jcs.92.1.67. [DOI] [PubMed] [Google Scholar]
  9. Grinnell F. Focal adhesion sites and the removal of substratum-bound fibronectin. J Cell Biol. 1986 Dec;103(6 Pt 2):2697–2706. doi: 10.1083/jcb.103.6.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hayman E. G., Pierschbacher M. D., Suzuki S., Ruoslahti E. Vitronectin--a major cell attachment-promoting protein in fetal bovine serum. Exp Cell Res. 1985 Oct;160(2):245–258. doi: 10.1016/0014-4827(85)90173-9. [DOI] [PubMed] [Google Scholar]
  11. Hughes R. C., Pena S. D., Clark J., Dourmashkin R. R. Molecular requirements for adhesion and spreading of hamster fibroblasts. Exp Cell Res. 1979 Jul;121(2):307–314. doi: 10.1016/0014-4827(79)90009-0. [DOI] [PubMed] [Google Scholar]
  12. Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. doi: 10.1083/jcb.103.6.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Humphries M. J. The molecular basis and specificity of integrin-ligand interactions. J Cell Sci. 1990 Dec;97(Pt 4):585–592. doi: 10.1242/jcs.97.4.585. [DOI] [PubMed] [Google Scholar]
  14. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  15. Hynes R. O., Yamada K. M. Fibronectins: multifunctional modular glycoproteins. J Cell Biol. 1982 Nov;95(2 Pt 1):369–377. doi: 10.1083/jcb.95.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Izzard C. S., Lochner L. R. Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. J Cell Sci. 1976 Jun;21(1):129–159. doi: 10.1242/jcs.21.1.129. [DOI] [PubMed] [Google Scholar]
  17. Juliano R. L. Membrane receptors for extracellular matrix macromolecules: relationship to cell adhesion and tumor metastasis. Biochim Biophys Acta. 1987 Nov 25;907(3):261–278. doi: 10.1016/0304-419x(87)90009-6. [DOI] [PubMed] [Google Scholar]
  18. Lindon J. N., McManama G., Kushner L., Merrill E. W., Salzman E. W. Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces? Blood. 1986 Aug;68(2):355–362. [PubMed] [Google Scholar]
  19. Massia S. P., Hubbell J. A. Covalent surface immobilization of Arg-Gly-Asp- and Tyr-Ile-Gly-Ser-Arg-containing peptides to obtain well-defined cell-adhesive substrates. Anal Biochem. 1990 Jun;187(2):292–301. doi: 10.1016/0003-2697(90)90459-m. [DOI] [PubMed] [Google Scholar]
  20. Pierschbacher M. D., Ruoslahti E. Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J Biol Chem. 1987 Dec 25;262(36):17294–17298. [PubMed] [Google Scholar]
  21. Ruoslahti E. Fibronectin and its receptors. Annu Rev Biochem. 1988;57:375–413. doi: 10.1146/annurev.bi.57.070188.002111. [DOI] [PubMed] [Google Scholar]
  22. Ruoslahti E. Integrins. J Clin Invest. 1991 Jan;87(1):1–5. doi: 10.1172/JCI114957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  24. Singer I. I., Kawka D. W., Scott S., Mumford R. A., Lark M. W. The fibronectin cell attachment sequence Arg-Gly-Asp-Ser promotes focal contact formation during early fibroblast attachment and spreading. J Cell Biol. 1987 Mar;104(3):573–584. doi: 10.1083/jcb.104.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Singer I. I., Scott S., Kawka D. W., Kazazis D. M., Gailit J., Ruoslahti E. Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation. J Cell Biol. 1988 Jun;106(6):2171–2182. doi: 10.1083/jcb.106.6.2171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Streeter H. B., Rees D. A. Fibroblast adhesion to RGDS shows novel features compared with fibronectin. J Cell Biol. 1987 Jul;105(1):507–515. doi: 10.1083/jcb.105.1.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tomasini B. R., Mosher D. F. Vitronectin. Prog Hemost Thromb. 1991;10:269–305. [PubMed] [Google Scholar]
  28. Underwood P. A., Bennett F. A. A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin. J Cell Sci. 1989 Aug;93(Pt 4):641–649. doi: 10.1242/jcs.93.4.641. [DOI] [PubMed] [Google Scholar]
  29. Woods A., Couchman J. R., Johansson S., Hök M. Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments. EMBO J. 1986 Apr;5(4):665–670. doi: 10.1002/j.1460-2075.1986.tb04265.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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