Role of Par3/Par6/aPKC Complex in Cell Polarity

描述(由申请人提供)： 上皮细胞形成高度极化的薄片，排列在许多内部器官中。在成人多囊肾病和急性缺血性肾损伤中，可观察到细胞极性的改变或明显的极性丧失。由Par3、Par6、非典型PKC组成的复合体(APKC)是极化的主要调节因子。莫斯托夫实验室使用三维胶原凝胶中的Madin-Darby犬肾(MDCK)细胞作为模型系统来研究细胞极性。在这种情况下，细胞形成由单层细胞构成的中空囊，所有单层细胞的顶面都面向中央管腔。这些研究的目的是了解Par3、Par6、aPKC和CDC42在极化囊泡结构发育中的作用。在极化的包囊中，Par3定位于紧密连接，而aPKC位于细胞的顶面。为了完成这些蛋白质在包囊形成过程中的定位研究，GFP标记的Par6将通过共聚焦显微镜进行定位。APKC/Par3/Par6复合体将通过免疫共沉淀法进一步表征。接下来，我们将研究在包囊形成和极性产生过程中，CDC42的亚细胞定位的变化。最后，我们将探讨Par3、Par6和aPKC在极性取向中的作用。这些蛋白质的显性负性突变体以及对照结构将在MDCK细胞中表达，这些蛋白质对三维培养的MDCK细胞的极性的影响将被检测。

DESCRIPTION (provided by applicant): Epithelial cells form highly polarized sheets that line many internal organs. In adult polycystic kidney disease and in acute ischemic renal injury, changes in cell polarity or frank loss of polarity are observed. A complex of Par3, Par6, atypical PKC (aPKC) is a master regulator of polarization. The Mostov lab has used Madin-Darby canine kidney (MDCK) cells in 3-dimensional collagen gels as a model system to study cell polarity. Under these conditions, the cells form hollow cysts lined by a monolayer of cells, all of which have the apical surface facing the central lumen. The goal of these studies is to understand the role of Par3, Par6, aPKC as well as Cdc42 in the development of polarized cyst structures. In polarized cysts, Par3 is localized to tight junctions, and aPKC is found on the apical surface of cells. To complete the localization studies of these proteins during cyst formation, a GFP-tagged Par6 will be localized by confocal microscopy. aPKC/Par3/Par6 complexes will be further characterized by co-immunoprecipitation studies. Next, changes in the subcellular localization of Cdc42 during cyst formation and the generation of polarity will be examined. Finally, the roles of Par3, Par6 and aPKC in orientation of polarity will be examined. Dominant negative mutants of each of these proteins, as well as control constructs will be expressed in MDCK cells, and the effects of these proteins on polarity in MDCK cells grown in 3 dimensional culture will be examined.