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Cellular responses to mechanical forces are crucial in embryonic development and adult physiology, and are involved in numerous diseases, including atherosclerosis, hypertension, osteoporosis, muscular dystrophy, myopathies and cancer. These responses are mediated by load-bearing subcellular structures, such as the plasma membrane, cell-adhesion complexes and the cytoskeleton. Recent work has demonstrated that these structures are dynamic, undergoing assembly, disassembly and movement, even when ostensibly stable. An emerging insight is that transduction of forces into biochemical signals occurs within the context of these processes. This framework helps to explain how forces of varying strengths or dynamic characteristics regulate distinct signalling pathways., There is growing recognition that mechanical factors, such as applied forces or the rigidity of the extracellular matrix (ECM), crucially influence the form and function of cells and organisms (1-3). [...]

Mechanical forces are central to developmental, physiological and pathological processes (1). However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy (2). The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent (3). We show that tension across vinculin in stable FAs is ~2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently., Focal adhesions (FAs) are complex intracellular linkages between integrins and the F-actin cytoskeleton that both transmit and respond to mechanical forces. FAs show complex mechanosensitivity such that they form or [...]