Spatiotemporal HO flashes coordinate actin cytoskeletal remodeling and regulate cell migration and wound healing.

Well-organized repair of damaged barrier epithelia is vital for infection control, resolution of inflammation, and enduring physical protection. Cysteine thiol and methionine oxidation are connected to cytoskeletal rearrangements in cell migration and wound healing, but how localized redox signaling is achieved to regulate dynamic processes remains elusive. Here, we identify DUOX2, a mucosal barrier NADPH oxidase, as vesicle-incorporated HO source, localizing to sites of cytoskeletal reorganization, and facilitating tunneling nanotube and lamellipodia formation. Using traceable fluorescent DUOX2 and the membrane-bound HO sensor HyPer7-MEM enabled insight into DUOX2 vesicle trafficking and HO generation at sites of actin polymerization and dynamic remodeling. Stable expression or ablation confirmed DUOX2 generated HO as a catalyst for cell-cell connections, random motility and directed migration. We identify a signaling axis from the mechanosensor PIEZO1 to DUOX2 and FER tyrosine kinase activation to initiate retraction wave-mediated efficient wound closure in epithelial cells, a prerequisite for barrier integrity.