VASP regulates the polar organization of adhesion-associated actin filaments

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. Although FA maturation and organization have been extensively studied, it remains unclear how regulatory proteins influence the 3D architecture of FAs. Here, we show that loss of the vasodilator-stimulated phosphoprotein (VASP) impairs adhesion dynamics. We employed CRISPR/Cas9-mediated knockout of VASP and/or the mechanosensitive adaptor protein zyxin to investigate their respective roles in actin–adhesion coupling. Loss of VASP and zyxin correlates with altered FA morphology and impaired dynamics. Using cryo-electron tomography (cryo-ET), we resolved the polarity of individual actin filaments associated with FAs and identified a contractility-related actin layer enriched with tropomyosin. VASP and zyxin are required for the assembly of dense and aligned actin bundles with uniform polarity, oriented with their barbed ends towards the cell edge. In contrast, the tropomyosin-decorated dorsal actin layer remains unaffected by these perturbations. Our findings reveal distinct, layered architectures within FAs and underscore the cooperative role of VASP and zyxin in stabilizing the organization of actin filaments at functional adhesion sites.