JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin

The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Call switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Call-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rabl3-binding protein).

紧密连接（TJs）的动态周转在上皮形态发生过程中的上皮 - 间充质转化和/或间充质 - 上皮转化中至关重要。我们先前证明Rab13特异性介导闭合蛋白的内吞循环。在此，我们鉴定出MICAL - L2（与CasL样蛋白2相互作用的分子）是一种新型的Rab13结合蛋白。免疫沉淀和免疫荧光显微镜显示，MICAL - L2通过其包含卷曲螺旋结构域的C末端特异性结合Rab13的GTP结合形式，并定位于上皮MTD - 1A细胞的紧密连接处。循环实验表明，缺失Rab13结合结构域的MICAL - L2突变体（MICAL - L2 - N）特异性抑制闭合蛋白的内吞循环，但不抑制转铁蛋白受体的循环。钙开关实验进一步揭示，MICAL - L2 - N以及Rab13 Q67L抑制闭合蛋白向质膜的募集、跨上皮电阻的产生以及细胞旁扩散屏障的形成。在肌动蛋白解聚时，MICAL - L2从紧密连接处移位，并分别沿着肌动蛋白解聚的MTD - 1A细胞中的辐射状肌动蛋白丝和成纤维细胞NIH3T3细胞中的应力纤维分布。这些结果表明，MICAL - L2通过将Rab13与肌动蛋白细胞骨架连接，介导闭合蛋白的内吞循环和功能性紧密连接的形成。我们将MICAL - L2重新命名为JRAB（连接Rab13结合蛋白）。