(86a) Measuring Mechanical Tension Across Vinculin Reveals Regulation of Focal Adhesion Dynamics

Mechanical forces are central to cell migration, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unraveling molecular mechanisms. Here we describe the development of an experimentally calibrated FRET-based biosensor that measures forces across specific proteins in cells with pico-Newton sensitivity. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is ~2.5pN 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.