Function of ArhGAP12 in the assembly and regulation of epithelial tight junctions

Abstract

In epithelia cells, tight junctions (TJ) form selective gates that control the paracellular diffusion of ions and solutes, and they act as a border between the apical and basolateral plasma-membrane domains. TJ formation and function are regulated by Rho-GTPases, whose activities are controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). We have identified ArhGAP12, a specific GAP for Rac1/Cdc42, as a novel TJ component recruited via ZO-2. Additionally, ArhGAP12 interacts with N-WASP and WAVE2, linking it to Arp2/3-driven actin polymerization at TJs. Our 3D cysts showed that the absence of ArhGAP12 impairs lumen formation. While our calcium-switch assay revealed that MDCK-II cells depleted of ArhGAP12 exhibited delayed TJ formation, accompanied by a delayed onset of TEER peaks. Permeability assays utilizing 4kDa FITC-dextran underscored ArhGAP12's critical role in regulating TJ permeability, as deletion of ArhGAP12 led to more restrictive leak pathways. Laser ablation experiments further revealed increased tension in ArhGAP12-deficient cells. Notably, both the restrictive leak pathways and heightened junctional tension in these cells were rescued upon inhibition of Arp2/3 activity. Together, these findings suggest that ArhGAP12 coordinates TJ formation and permeability, potentially by regulating N-WASP and WAVE2-mediated actin assembly.