Abstract
Focal adhesions (FAs) are dynamic structures central to cell migration, serving as mechanotransduction sites linking the extracellular matrix (ECM) to intracellular signaling pathways such as FA kinase (FAK). How FAK becomes activated in response to cell-ECM adhesive forces at single FAs to facilitate directional motion is poorly understood. Using micropillar-based force microscopy and FA-targeted FRET biosensors, we monitored real-time traction forces and FAK activity at individual FAs during assembly and disassembly. Our results demonstrate oscillatory temporal coupling of traction force and FAK activity in high-tension FAs before FA disassembly. Cross-correlation analyses revealed that force precedes FAK activation, guiding FA turnover. Atomistic molecular simulations unveiled a force-induced mechanism where traction forces disrupt autoinhibitory FERM-kinase interactions in FAK, enabling catalytic activity without structural unfolding. Our findings provide mechanistic insights into the spatiotemporal integration of mechanical forces and biochemical signaling in cell migration.