Position-dependent linkages of fibronectin-integrin-cytoskeleton

Position-dependent cycling of integrin interactions with both the cytoskele ton and extracellular matrix (ECM) is essential for cell spreading, migrati on, and wound healing. Whether there are regional changes in integrin conce ntration, ligand affinity or cytoskeleton crosslinking of liganded integrin s has been unclear. Here, we directly demonstrate a position-dependent bind ing and release cycle of fibronectin-integrin-cytoskeleton interactions wit h preferential binding at the front of motile 3T3 fibroblasts and release a t the endoplasm-ectoplasm boundary. Polystyrene beads coated with low conce ntrations of an integrin-binding fragment of fibronectin (fibronectin type III domains 7-10) were 3-4 times more likely to bind to integrins when plac ed within 0.5 microns vs, 0.5-3 microns from the leading edge. Integrins we re not concentrated at the leading edge, nor did anti-integrin antibody-coa ted beads bind preferentially at the leading edge. However, diffusing ligan ded integrins attached to the cytoskeleton preferentially at the leading ed ge, Cytochalasin inhibited edge binding, which suggested that cytoskeleton binding to the integrins could alter the avidity for ligand beads. Further, at the ectoplasm-endoplasm boundary, the velocity of bead movement decreas ed, diffusive motion increased, and approximately one-third of the beads we re released into the medium. We suggest that cytoskeleton linkage of ligand ed integrins stabilizes integrin-ECM bonds at the front whereas release of cytoskeleton-integrin links weakens integrin-ECM bonds at the back of lamel lipodia.