Benais-Pont Gaelle, Punn Anu, Flores-Maldonado Catalina, Eckert Judith, Raposo Graca, Fleming Tom P, Cereijido Marcelino, Balda Maria S, Matter Karl
Department of Cell Biology, University of Geneva, 1211 Geneva, Switzerland.
J Cell Biol. 2003 Mar 3;160(5):729-40. doi: 10.1083/jcb.200211047. Epub 2003 Feb 25.
Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.
Rho家族GTP酶是上皮紧密连接（TJ）的重要调节因子；然而，关于GTP酶在TJ组装和功能过程中自身是如何被调控的，我们却知之甚少。我们已经鉴定并克隆了一种原癌基因Dbl家族的犬鸟嘌呤核苷酸交换因子（GEF），它能激活Rho并与TJ相关联。基于序列相似性搜索以及免疫和功能数据，这种蛋白质是人类GEF-H1和小鼠Lfc的犬类同源物，这两种先前鉴定出的Rho特异性交换因子已知在非极化细胞中与微管相关联。与这些观察结果一致，增殖的MDCK细胞的免疫荧光显示内源性犬类GEF-H1/Lfc与有丝分裂纺锤体相关联。基于在极化MDCK细胞中的过表达和RNA干扰的功能分析表明，这种Rho的交换因子调节小亲水性示踪剂的细胞旁通透性。尽管过表达导致大小选择性细胞旁通透性增加，但通过测量跨上皮电阻、免疫荧光和冷冻断裂分析确定，这类细胞系呈现正常的整体形态并形成完全组装的TJ。这些数据表明GEF-H1/Lfc是TJ的一个组成部分，并在上皮通透性调节中发挥作用。
