Force at Early Adhesion Formation: Integrin Activation Triggers Contractile Movement and Subsequent Outward Translocation of Integrin-RGD Clusters

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Integrin-mediated adhesions play important roles in many biology events, including stem cell differentiation, cancer metastasis, and the immune response. However, The mechanism of early adhesion formation remains unclear. Using mobile RGD ligands on lipid bilayers with nano-fabricated physical barriers, we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. At first, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin and focal adhesion kinase, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Src-dependent actin polymerization on integrin clusters and subsequent contraction stimulate active spreading with outward forces from actin polymerization followed by a second wave of contraction. These early mechanical steps provide new targets to control integrin-dependent adhesion and motility.