Involvement of actinin-4 in the recruitment of JRAB/MICAL-L2 to cell-cell junctions and the formation of functional tight junctions
Involvement of actinin-4 in the recruitment of JRAB/MICAL-L2 to cell-cell junctions and the formation of functional tight junctions
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DOI：
10.1128/mcb.00144-08
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发表时间：
2008-05-01
影响因子：
5.3
通讯作者：
Sasaki, Takuya
中科院分区：
文献类型：
--
作者：
Nakatsuji, Hiroyoshi;Nishimura, Noriyuki;Sasaki, Takuya
紧密连接（TJs）是经历连续重塑的细胞-细胞粘附结构。我们以前证明，Rab 13和连接Rab 13结合蛋白（JRAB）/分子与CasL样2（MICAL-L2）定位在TJ和介导的内吞循环的完整TJ蛋白occludin和功能性TJ的形成。在这里，我们研究了JRAB/MICAL-L2如何针对TJ。使用一系列缺失突变体，我们发现了JRAB/MICAL-L2中的质膜（PM）靶向结构域。然后，我们使用酵母双杂交系统将最初作为与细胞运动性和癌症侵袭/转移相关的肌动蛋白结合蛋白分离的肌动蛋白-4鉴定为JRAB/MICAL-L2的PM靶向结构域的结合蛋白。肌动蛋白-4与JRAB/MICAL-L2共定位于细胞-细胞连接处，并将JRAB/MICAL-L2连接至F-肌动蛋白。虽然辅肌动蛋白-4结合JRAB/MICAL-L2没有Rab 13，辅肌动蛋白-4-JRAB/MICAL-L2的相互作用增强Rab 13激活。消耗辅肌动蛋白-4通过使用小干扰RNA抑制招聘occludin的TJ在Ca 2+开关。在再铺板后的上皮极化期间，JRAB/MICAL-L2从细胞质募集到细胞-细胞连接处。这种JRAB/MICAL-L2的募集以及功能性TJ的形成在辅肌动蛋白-4耗尽的细胞中被延迟。这些结果表明，辅肌动蛋白-4参与募集JRAB/MICAL-L2到细胞-细胞连接和形成功能性TJ。
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 Ca2+ 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.