VASP cooperates with zyxin to organize mechanically competent adhesions

Abstract Focal adhesions (FAs) are dynamic macromolecular assemblies that anchor the actin cytoskeleton to the extracellular matrix via integrin receptors, thereby regulating cell morphology and migration. Here, we show that the vasodilator-stimulated phosphoprotein (VASP) cooperates with the mechanosensitive scaffold protein zyxin to maintain FA during maturation under actomyosin-generated tension. Using sequential CRISPR/Cas9-mediated gene editing and cryo-electron tomography, we resolved the in situ nanoarchitecture of the FA-stress fiber interface. We establish that tropomyosin decorates the dorsal aspect of adhesion-associated stress fibers independently of VASP and zyxin. VASP and zyxin are required for the assembly of dense, aligned actin bundles with uniform polarity, oriented with their barbed ends toward the cell edge. Live-cell imaging and quantitative analyses of cells confined on micropatterns reveal that VASP and zyxin cooperate to preserve the mechanical coupling between FAs and the actin cytoskeleton under contractile load. Our findings establish that VASP and zyxin perform distinct yet complementary functions to organize FA-associated actin into mechanically competent force-transmitting units.