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，排列在许多内脏器官上。在成人多囊肾病和急性缺血性肾损伤中，观察到细胞极性改变或极性明显丧失。Par 3、Par 6、非典型PKC（aPKC）的复合物是极化的主要调节剂。Mostov实验室使用Madin-Darby犬肾（MDCK）细胞在三维胶原凝胶中作为模型系统来研究细胞极性。 在这些条件下，细胞形成由单层细胞内衬的中空囊，所有细胞的顶面都面向中央腔。这些研究的目的是了解Par 3，Par 6，aPKC以及Cdc 42在极化囊肿结构发育中的作用。在极化囊肿中，Par 3定位于紧密连接，aPKC发现于细胞的顶端表面。为了完成这些蛋白质在囊肿形成过程中的定位研究，将通过共聚焦显微镜定位GFP标记的Par 6。 aPKC/Par 3/Par 6复合物将通过免疫共沉淀研究进一步表征。接下来，将检查Cdc 42在囊肿形成和极性产生期间的亚细胞定位的变化。 最后，将检查Par 3、Par 6和aPKC在极性取向中的作用。将在MDCK细胞中表达这些蛋白质中的每一种的显性阴性突变体以及对照构建体，并将检查这些蛋白质对在三维培养物中生长的MDCK细胞中的极性的影响。

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.