ARHGAP12 suppresses F-actin assembly to control epithelial tight junction mechanics and paracellular leak pathway permeability

Tight junctions (TJ) separate body compartments and control the paracellular transport of ions, solutes, and macromolecules across epithelial barriers. There is evidence that claudin-based ion transport (the pore pathway) and the paracellular transport of macromolecules (the leak pathway) are independently regulated processes. However, how leak pathway permeability is controlled is not well understood. Here we have identified the Cdc42/Rac GTPase activating protein ARHGAP12 as a novel and specific regulator of the leak pathway. ARHGAP12 is recruited to TJ via an interaction between its SH3 domain and the TJ protein ZO-2. Using a combination of biochemical and biophysical approaches, in vitro actin polymerisation assays, and permeability assays in MDCK-II cells, we show that ARHGAP12 suppresses N-WASP-mediated F-actin assembly at TJ to dampen junctional tension. This promotes paracellular leak pathway permeability without affecting ion flux. Mechanistically, we demonstrate that the ARHGAP12 tandem WW domain interacts directly and in a multivalent manner with an array of PPxR motifs in the proline-rich domain of N-WASP. This interaction is sufficient to suppress SH3 domain-mediated N-WASP oligomerisation and Arp2/3-driven F-actin assembly in vitro. Collectively our data demonstrate a critical role for ARHGAP12 in suppressing junctional F-actin assembly and tension to promote the flux of small macromolecules across the TJ.