Guillemot Laurent, Guerrera Diego, Spadaro Domenica, Tapia Rocio, Jond Lionel, Citi Sandra
Department of Molecular Biology, University of Geneva, CH-1211 Geneva, Switzerland.
Department of Molecular Biology, University of Geneva, CH-1211 Geneva, SwitzerlandDepartment of Cell Biology, University of Geneva, CH-1211 Geneva, SwitzerlandInstitute of Genetics and Genomics in Geneva, University of Geneva, CH-1211 Geneva, Switzerland
Mol Biol Cell. 2014 Jul 1;25(13):1995-2005. doi: 10.1091/mbc.E13-11-0680. Epub 2014 May 7.
The regulation of Rho-family GTPases is crucial to direct the formation of cell-cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin-Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.
Rho家族GTP酶的调节对于指导细胞间连接和组织屏障的形成至关重要。cingulin（CGN）和paracingulin（CGNL1）通过与RhoA鸟苷酸交换因子相互作用来控制上皮细胞中的RhoA激活。CGNL1的缺失也会在连接组装过程中抑制Rac1激活。在这里，我们意外地发现，尽管Rac1激活剂Tiam1的连接募集减少，但同时缺失CGN和CGNL1的Madin-Darby犬肾上皮细胞（双敲除细胞）仍表现出正常的Rac1激活和紧密连接（TJ）形成。双敲除细胞中Rac1抑制剂MgcRacGAP的表达降低，并且通过外源表达MgcRacGAP可以挽救屏障发育和Rac1激活表型。MgcRacGAP与CGN和CGNL1在紧密连接处共定位，形成复合物，并在体外与CGN和CGNL1直接相互作用。上皮细胞中CGN或CGNL1的缺失导致MgcRacGAP的连接定位降低，但不影响ECT2（一种与中心纺锤体相互作用的Rho鸟苷酸交换因子）的连接定位。这些结果为连接处Rho家族GTP酶活性的协调提供了新的见解，因为在连接成熟过程中CGN和CGNL1在紧密连接处的顶端积累提供了一种机制，通过募集MgcRacGAP在空间上限制Rac1激活的下调。
