Piezo1 balances focal and reticular adhesions to enable EGFR clathrin-mediated endocytosis

Cells attach to the extracellular matrix through distinct integrin-mediated adhesive structures, including force-transmitting focal adhesions (FAs) and clathrin-enriched reticular adhesions (RAs). FAs enable mesenchymal cell migration and disassemble at mitotic entry, whereas RAs impede migration, persist during mitosis, and contribute to clathrin-mediated endocytosis (CME) as they disassemble. FAs grow with RhoA contractility, whereas RAs shrink, but the mechanisms coordinating these opposing responses remain unclear. Here, we identify the mechanically activated ion channel Piezo1 as a master regulator of FA/RA balance. Piezo1-dependent calcium influx activates the Src family kinase Fyn, which activates two actin polymerization pathways: FA and stress fiber growth via VAV2-RhoA and RA disassembly via N-WASP-Arp2/3. Inhibition or knockdown of Piezo1, Fyn, or VAV2 decreases FA size and increases RA coverage. Critically, cells lacking Piezo1 fail to internalize ligand-activated EGFR on stiff substrates despite normal CME on soft substrates, establishing an essential role for Piezo1 in EGFR CME mechanoadaption. Our findings reveal Piezo1 as the mechanosensor linking membrane tension to coordinated actin polymerization pathways that co-regulate cell-matrix adhesion and endocytosis. Given that CME contributes to viral entry into host cells and cancer resistance to anti-EGFR antibody therapy, targeting the Piezo1-RA-CME axis may offer novel therapeutic opportunities.