Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing 1, 2. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling1 -3. Focal adhesions consist of a complex network 4 of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque5 , 6 linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine7 ,8 . However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) 9 to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ~40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino-and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.Modern understanding of cellular function is founded on the revolution in the 1950s to 1970s in visualizing cellular ultrastructure by electron microscopy 10,11 . Together with the

Cell migration initiates by extension of the actin cytoskeleton at the leading edge. Computational analysis of fluorescent speckle microscopy movies of migrating epithelial cells revealed this process is mediated by two spatially colocalized but kinematically, kinetically, molecularly, and functionally distinct actin networks. A lamellipodium network assembled at the leading edge but completely disassembled within 1 to 3 micrometers. It was weakly coupled to the rest of the cytoskeleton and promoted the random protrusion and retraction of the leading edge. Productive cell advance was a function of the second colocalized network, the lamella, where actomyosin contraction was integrated with substrate adhesion.

Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

Summary Cell migration toward areas of higher extracellular matrix (ECM) rigidity via a process called “durotaxis” is thought to contribute to development, immune response, and cancer metastasis. To understand how cells sample ECM rigidity to guide durotaxis, we characterized cell-generated forces on the nanoscale within single mature integrin-based focal adhesions (FAs). We found that individual FAs act autonomously, exhibiting either stable or dynamically fluctuating (“tugging”) traction. We show that a FAK/phosphopaxillin/vinculin pathway is essential for high FA traction and to enable tugging FA traction over a broad range of ECM rigidities. We show that tugging FA traction is dispensable for FA maturation, chemotaxis, and haptotaxis but is critical to direct cell migration toward rigid ECM. We conclude that individual FAs dynamically sample rigidity by applying fluctuating pulling forces to the ECM to act as sensors to guide durotaxis, and that FAK/phosphopaxillin/vinculin signaling defines the rigidity range over which this dynamic sensing process operates.