An RGD spacing of 440 nm is sufficient for integrin α_vβ₃-mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation

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 10₄ 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 10⁴ 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 avft blocked cell adhesion and spreading on substrates containing 100 fmol/cm² of surface-bound GRGDY, while fibronectin receptor antiserum specific for α₅β₁ did not. Furthermore, α_vβ₃ was observed to cluster into focal contacts in spread cells, but α₅β₁ did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for α_vβ₃-mediated cellular spreading, while 140 nm was required for α_vβ₃-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.