Sumida Grant M, Yamada Soichiro
From the Department of Biomedical Engineering, University of California, Davis, California 95616.
From the Department of Biomedical Engineering, University of California, Davis, California 95616
J Biol Chem. 2015 Feb 6;290(6):3238-47. doi: 10.1074/jbc.M114.612168. Epub 2014 Dec 19.
Actin regulation is required for membrane activities that drive cell adhesion and migration. The Rho GTPase family plays critical roles in actin and membrane dynamics; however, the roles of the Rho GTPase family are not limited to cell adhesion and migration. Using micron-sized obstacles to induce the formation of self-contacts in epithelial cells, we previously showed that self-adhesion is distinct from cell-to-cell adhesion in that self-contacts are eliminated by membrane fusion. In the current study, we identified Rho GTPases, RhoA, Rac1, and Cdc42, as potential upstream regulators of membrane fusion. The RhoA downstream effector myosin II is required for fusion as the expression of mutant myosin light chain reduced membrane fusion. Furthermore, an inhibitor of the Arp2/3 complex, a downstream effector of Rac1 and Cdc42, also reduced self-contact-induced membrane fusion. At self-contacts, while the concentration of E-cadherin diminished, the intensity of GFP-tagged Arp3 rapidly fluctuated then decreased and stabilized after membrane fusion. Taken together, these data suggest that the Arp2/3 complex-mediated actin polymerization brings two opposing membranes into close apposition by possibly excluding E-cadherin from contact sites, thus promoting membrane fusion at self-contacts.
驱动细胞黏附和迁移的膜活动需要肌动蛋白调节。Rho GTP酶家族在肌动蛋白和膜动力学中起关键作用；然而，Rho GTP酶家族的作用并不局限于细胞黏附和迁移。我们之前利用微米级障碍物诱导上皮细胞形成自接触，结果表明自黏附与细胞间黏附不同，自接触可通过膜融合消除。在当前研究中，我们确定Rho GTP酶RhoA、Rac1和Cdc42是膜融合的潜在上游调节因子。RhoA的下游效应器肌球蛋白II是融合所必需的，因为突变型肌球蛋白轻链的表达降低了膜融合。此外，Rac1和Cdc42的下游效应器Arp2/3复合物的抑制剂也降低了自接触诱导的膜融合。在自接触处，虽然E-钙黏蛋白的浓度降低，但绿色荧光蛋白标记的Arp3的强度迅速波动，然后在膜融合后下降并稳定。综上所述，这些数据表明，Arp2/3复合物介导的肌动蛋白聚合可能通过将E-钙黏蛋白排除在接触位点之外，使两个相对的膜紧密靠近，从而促进自接触处的膜融合。
