SPATIOTEMPORAL COORDINATION OF THE ACTIN CYTOSKELETON AND INTEGRIN ADHESION

Integrin-based adhesions mediate critical interactions between the cell and its external environment. Integrins assemble into macromolecular "focal adhesions" (FAs) that contain hundreds of proteins and indirectly connect integrin cytoplasmic tails to the actin cytoskeleton. Forces transmitted across FAs drive tissue morphogenesis, cell movement, and extracellular matrix (ECM) remodeling. During cell migration, actin polymerization drives protrusion of the leading edge and integrins mediate adhesion to the ECM. However, efficient movement requires that these distinct processes are spatiotemporally coordinated. Here we provide evidence that Arp2/3-mediated actin polymerization can stimulate integrin adhesion throughout the cell surface, while vinculin activation is spatiotemporally controlled by the macromolecular FA architecture to engage actin retrograde flow to growing FAs. We have identified and characterized "adhesive F-actin waves," a novel integrin-mediated adhesion complex coupled to ventral actin polymerization. Integrins engage the ECM downstream of Arp2/3-mediated ventral F-actin waves in a variety of mammalian cell types. These adhesive F-actin waves require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. These results suggest that Arp2/3 activity, rather than the specific lamellipodium structure, is important for initiating integrin adhesion. Vinculin is recruited to FAs as they grow and compositionally mature to engage actin retrograde flow and mechanically reinforce the integrin-actin linkage. Vinculin has many binding partners at FAs, and its interactions are regulated by an auto-inhibitory, high-affinity intramolecular interaction. To study the spatiotemporal regulation of vinculin activation and vinculin function, we used superresolution microscopy to assay vinculin nanoscale organization and a FRET biosensor to assay vinculin conformation. We found that movement up the laminar FA structure during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin is recruited to the lower integrin signaling layer by binding to phospho-paxillin. Talin and actin binding activates vinculin, and talin targets active vinculin to the higher FA layers where vinculin can engage actin retrograde flow. Thus, specific protein interactions occur within distinct FA nano-domains to regulate vinculin recruitment, activation, and function in during FA maturation.Doctor of Philosoph