Focal adhesions (FAs) are macromolecular complexes that regulate cell adhesion and mechanotransduction. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoactivation (FLAP), we found that the mobility of core FA proteins correlates with protein function. Structural proteins such as tensin, talin and vinculin are significantly less mobile in FAs than signaling proteins such as FAK and paxillin. The mobilities of the structural proteins are directly influenced by substrate stiffness, suggesting they are involved in sensing the rigidity of the extracellular environment. The turnover rates of FAK and paxillin as well as kindlin2 are not influenced by substrate stiffness. By using specific Src and FAK kinase inhibitors, we reveal that force-sensing by vinculin occurs independently of FAK and paxillin phosphorylation. However, this phosphorylation is required for downstream, Rac1-driven cellular processes, such as protrusion and cell migration. Overall, we show that the FA is composed of different functional modules that separately control mechanosensing and the cellular mechano-response.