The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s⁻¹. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The ‘allosteric vinculin mutant’ is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

The interface between the cellular actin network and diverse forms of integrin-mediated cell adhesions displays a unique capacity to serve as accurate chemical and mechanical sensors of the cell’s microenvironment. Focal adhesion-like structures of diverse cell types, podosomes in osteoclasts, and invadopodia of invading cancer cells display distinct morphologies and apparent functions. Yet, all three share a similar composition and mode of coupling between a protrusive structure (the lamellipodium, the core actin bundle of the podosome, and the invadopodia protrusion, respectively), and a nearby adhesion site. Cytoskeletal or external forces, applied to the adhesion sites, trigger a cascade of unfolding and activation of key adhesome components (e.g., talin, vinculin, integrin), which in turn, trigger the assembly of adhesion sites and generation of adhesion-mediated signals that affect cell behavior and fate. The structural and molecular mechanisms underlying the dynamic crosstalk between the actin cytoskeleton and the adhesome network are discussed.

High‐content image‐based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high‐quality open‐access data sharing and meta‐analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta‐analysis of results from live‐cell microscopy, have not been systematically investigated. Here, using high‐content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta‐analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image‐based datasets of perturbation experiments. Thus, reproducible quantitative high‐content cell image analysis of perturbation effects and meta‐analysis depend on standardized procedures combined with batch correction.

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.

Summary: The molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro , possibly accounting for their scarcity in vivo . These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits precursor cell fusion, and for which SNX10 is required.