Tight Junction Proteins and Epithelial Polarity

描述（申请人提供）：适当的顶基底上皮极性对正常肾功能至关重要，在急性肾小管坏死和多囊肾病等肾脏疾病中受到干扰。最近的研究进展为多蛋白复合物在上皮细胞极化和蛋白靶向中的作用提供了新的见解。在目前的资助期内，我们的实验室发现了定位于紧密连接的支架蛋白，对细胞极化至关重要。我们的工作主要集中在与PDZ小结构域蛋白mLin-7相关的蛋白质上。我们证明了这些mLin-7结合伙伴之一，称为蛋白相关的Lin-7 1 （PALS1）定位于紧密连接和复合物与PALS1相关的紧密连接蛋白（PATJ）和crumbs。PALS1和PATJ是包含PDZ和L27结构域的支架蛋白，而crumss是一个小的顶端跨膜蛋白。利用siRNA和显性阴性蛋白，我们证明了PALS1和PATJ对上皮细胞极性至关重要。我们还发现PALS1直接与由三种蛋白Par3/Par6/非典型蛋白激酶c组成的共同极性盒相互作用。我们研究的目标是了解PALS1和PATJ如何控制上皮细胞极化的初始步骤。我们假设支架蛋白，PALS1和PATJ从细胞内的位置移动到极化上皮细胞的一个点，指导紧密连接定位和从根尖到基底侧膜表面的过渡。本研究的目的是了解这些蛋白是如何在极化上皮细胞中识别这一点的，以及它们是如何招募参与细胞极化的其他蛋白的。为了实现这些目标，我们将研究在MDCK细胞内控制其运动的PALS1和PATJ结构域。我们还将研究这些蛋白质的哪些结构域在定位、运输、紧密连接形成和细胞极化中是重要的。此外，我们将研究控制这些事件的信号转导过程。这些研究将揭示早期上皮极化的必要过程，并对健康和疾病中的肾上皮功能具有重要意义。

DESCRIPTION (provided by applicant): Proper apico-basal epithelial polarity is crucial for normal kidney function and is perturbed in kidney diseases such as acute tubular necrosis and polycystic kidney disease. Recent advances by our group and others have provided new insights into the role of multi-protein complexes in epithelial cell polarization and protein targeting. In the current funding period our laboratory identified scaffolding proteins that localize to the tight junction and are crucial for cell polarization. Our work has focused on proteins associated with the small PDZ domain protein, mLin-7. We demonstrated that one of these mLin-7 binding partners called Protein Associated with Lin Seven 1 (PALS1) localizes to tight junctions and complexes with PALS1 Associated Tight Junction Protein (PATJ) and CrumbsS. PALS1 and PATJ are scaffolding proteins that contain PDZ and L27 domains while CrumbsS is a small apical transmembrane protein. Using siRNA and dominant negative proteins we have demonstrated that PALS1 and PATJ are crucial for epithelial cell polarity. We have also found that PALS1 directly interacts with a common polarity cassette consisting of three proteins Par3/Par6/atypical Protein Kinase C. The goal of our research is to understand how PALS1 and PATJ control the initial steps in epithelial cell polarization. We hypothesize that the scaffolding proteins, PALS1 and PATJ move from an intracellular location to mark a spot in polarizing epithelial cells that directs tight junction localization and the transition from apical to basolateral membrane surfaces. The goals of this proposal are to understand how these proteins come to identify this spot in polarizing epithelial cells and how they proceed to recruit other proteins involved in cell polarization. To achieve these goals we will examine the domains of PALS1 AND PATJ that control their movement within MDCK cells. We will also examine which domains of these proteins are important in localization, trafficking, tight junction formation and cell polarization. In addition, we will examine the signal transduction processes that control these events. These studies will shed new light on the processes necessary for early epithelial polarization and have important implication for renal epithelial function in health and disease.