SUMMARY
Cell migration is crucial for development and tissue homeostasis, while its dysregulation leads to severe pathologies. Cell migration is driven by the extension of actin-based lamellipodia protrusions, powered by actin polymerization, which is tightly regulated by signaling pathways, including Rho GTPases and Ca2+signaling. While the importance of Ca2+signaling in lamellipodia protrusions has been established, the molecular mechanisms linking Ca2+to lamellipodia assembly are unknown. Here, we identify a novel Ca2+signaling axis involving the mechano-gated channel TRPV4, which regulates lamellipodia protrusions in various cell types. Using Ca2+and FRET imaging, we demonstrate that TRPV4-mediated Ca2+influx upregulates RhoA activity within lamellipodia, which then facilitates formin-mediated actin assembly. Mechanistically, we identify CaMKII and TEM4 as key mediators relaying the TRPV4-mediated Ca2+signal to RhoA. These data define a molecular pathway by which Ca2+influx regulates small GTPase activity within a specific cellular domain – lamellipodia - and demonstrate the critical role in organizing the actin machinery and promoting cell migration in diverse biological contexts.