Abstract
Tight junctions (TJs) are cell-cell adhesive structures that undergo continuous remodeling. We previously demonstrated that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) localized at TJs and mediated the endocytic recycling of the integral TJ protein occludin and the formation of functional TJs. Here, we investigated how JRAB/MICAL-L2 was targeted to TJs. Using a series of deletion mutants, we found the plasma membrane (PM)-targeting domain within JRAB/MICAL-L2. We then identified actinin-4, which was originally isolated as an actin-binding protein associated with cell motility and cancer invasion/metastasis, as a binding protein for the PM-targeting domain of JRAB/MICAL-L2, using a yeast two-hybrid system. Actinin-4 was colocalized with JRAB/MICAL-L2 at cell-cell junctions and linked JRAB/MICAL-L2 to F-actin. Although actinin-4 bound to JRAB/MICAL-L2 without Rab13, the actinin-4-JRAB/MICAL-L2 interaction was enhanced by Rab13 activation. Depletion of actinin-4 by using small interfering RNA inhibited the recruitment of occludin to TJs during the Ca(2+) switch. During the epithelial polarization after replating, JRAB/MICAL-L2 was recruited from the cytosol to cell-cell junctions. This JRAB/MICAL-L2 recruitment as well as the formation of functional TJs was delayed in actinin-4-depleted cells. These results indicate that actinin-4 is involved in recruiting JRAB/MICAL-L2 to cell-cell junctions and forming functional TJs.