Significance: Cells can sense their environment by using hair-like structures called filopodia that often exert pulling forces upon adhesive tip contact. We show, using optical tweezers and confocal microscopy, that the retraction force is generated by the dynamics of the cortical actin cytoskeleton, constantly pulling on the filopodial base. The weakest point of force transduction is at the tip between the actin shaft and the membrane. This allows tip-bound filopodia to apply controlled forces and to use a “load-and-fail” sensing process.

The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP. Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments. VASP increases the velocity of beads. VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells. The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins. However, VASP does not compete with capping proteins for binding barbed ends of actin filaments. VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria. VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching.

A cell constantly adapts to its environment. Cell decisions to survive, to proliferate or to migrate are dictated not only by soluble growth factors, but also through the direct interaction of the cell with the surrounding extracellular matrix (ECM). Integrins and their connections to the actin cytoskeleton are crucial for monitoring cell attachment and the physical properties of the substratum. Cell adhesion dynamics are modulated in complex ways by the polymerization of branched and linear actin arrays, which in turn reinforce ECM-cytoskeleton connection. This review describes the major actin regulators, Ena/VASP proteins, formins and Arp2/3 complexes, in the context of signaling pathways downstream of integrins. We focus on the specific signaling pathways that transduce the rigidity of the substrate and which control durotaxis, i.e. directed migration of cells towards increased ECM rigidity. By doing so, we highlight several recent findings on mechanotransduction and put them into a broad integrative perspective that is the result of decades of intense research on the actin cytoskeleton and its regulation.

Twinfilins are conserved actin‐binding proteins composed of two actin depolymerizing factor homology (ADF‐H) domains. Twinfilins are involved in diverse morphological and motile processes, but their mechanism of action has not been elucidated. Here, we show that mammalian twinfilin both sequesters ADP‐G‐actin and caps filament barbed ends with preferential affinity for ADP‐bound ends. Twinfilin replaces capping protein and promotes motility of N‐WASP functionalized beads in a biomimetic motility assay, indicating that the capping activity supports twinfilin's function in motility. Consistently, in vivo twinfilin localizes to actin tails of propelling endosomes. The ADP‐actin‐sequestering activity cooperates with the filament capping activity of twinfilin to finely regulate motility due to processive filament assembly catalyzed by formin‐functionalized beads. The isolated ADF‐H domains do not cap barbed ends nor promote motility, but sequester ADP‐actin, the C‐terminal domain showing the highest affinity. A structural model for binding of twinfilin to barbed ends is proposed based on the similar foldings of twinfilin ADF‐H domains and gelsolin segments.