Paschoud Serge, Yu Dan, Pulimeno Pamela, Jond Lionel, Turner Jerrold R, Citi Sandra
Department of Molecular Biology, University of Geneva, Geneva, Switzerland.
Mol Membr Biol. 2011 Feb;28(2):123-35. doi: 10.3109/09687688.2010.538937. Epub 2010 Dec 17.
Cingulin (CGN) and paracingulin (CGNL1) are structurally related proteins that regulate Rho family GTPases by recruiting guanine nucleotide exchange factors to epithelial junctions. Although the subcellular localization of cingulin and paracingulin is likely to be essential for their role as adaptor proteins, nothing is known on their in vivo localization, and their dynamics of exchange with the junctional membrane. To address these questions, we generated stable clones of MDCK cells expressing fluorescently tagged cingulin and paracingulin. By FRAP analysis, cingulin and paracingulin show a very similar dynamic behaviour, with recovery curves and mobile fractions that are distinct from ZO-1, and indicate a rapid exchange with a cytosolic pool. Interestingly, only paracingulin, but not cingulin, is peripherally localized in isolated cells, requires the integrity of the microtubule cytoskeleton to be stably anchored to junctions, and associates with E-cadherin. In contrast, both proteins require the integrity of the actin cytoskeleton to maintain their junctional localization. Although cingulin and paracingulin form a complex and can interact in vitro, the junctional recruitment and the dynamics of membrane exchange of paracingulin is independent of cingulin, and vice-versa. In summary, cingulin and paracingulin show a similar dynamic behaviour, but partially distinct localizations and functional interactions with the cytoskeleton, and are recruited independently to junctions.
cingulin（CGN）和副cingulin（CGNL1）是结构相关的蛋白质，它们通过将鸟嘌呤核苷酸交换因子招募到上皮连接来调节Rho家族GTP酶。尽管cingulin和副cingulin的亚细胞定位可能对其作为衔接蛋白的作用至关重要，但关于它们在体内的定位以及与连接膜的交换动态却一无所知。为了解决这些问题，我们构建了表达荧光标记的cingulin和副cingulin的MDCK细胞稳定克隆。通过荧光漂白恢复（FRAP）分析，cingulin和副cingulin表现出非常相似的动态行为，其恢复曲线和移动分数与ZO-1不同，表明它们与胞质池快速交换。有趣的是，只有副cingulin而不是cingulin在外周定位在分离的细胞中，需要微管细胞骨架的完整性才能稳定地锚定在连接处，并与E-钙黏着蛋白结合。相比之下，两种蛋白质都需要肌动蛋白细胞骨架的完整性来维持其连接处的定位。尽管cingulin和副cingulin形成复合物并能在体外相互作用，但副cingulin的连接处募集和膜交换动态独立于cingulin，反之亦然。总之，cingulin和副cingulin表现出相似的动态行为，但在与细胞骨架的定位和功能相互作用方面部分不同，并且独立地募集到连接处。
