Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading

Integrin receptors, and associated cytoplasmic proteins mediate adhesion, cell signaling and connections to the cytoskeleton. Using fluorescent protein chimeras, we analyzed initial integrin adhesion in spreading fibroblasts with Total Internal Reflection Fluorescence (TIRF) microscopy. Surprisingly, sequential radial projection of integrin and actin containing filopodia formed the initial cell-matrix contacts. These Cdc42-dependent, integrin-containing projections recruited cytoplasmic focal adhesion (FA) proteins in a hierarchical manner; initially talin with integrin and subsequently FAK and paxillin. Radial FA structures then anchored cortical actin bridges between them and subsequently cells reorganized their actin, a process promoted by Src, and characterized by lateral FA reorientation to provide anchor points for actin stress fibers. Finally, the nascent adhesions coalesced until they formed mature FAs.