Molecular Mechanisms of Polarized Endocytic Traffic in Epithelial Cells

描述（由申请人提供）：肾和肠上皮细胞在结构和功能上极化以运输特定分子，同时维持跨上皮屏障。这种细胞不对称对于上皮组织的正常功能至关重要，并依赖于极化的内吞运输途径。此外，上皮细胞与邻近细胞协调其极化形成顶管腔，这是肾脏和肠道结构建立的关键步骤，从而发挥功能。事实上，极化上皮运输和根尖管腔形成的功能障碍是多种肾脏和肠道疾病的原因。本研究旨在填补我们对上皮细胞生物学理解的关键空白，即在肾脏形态发生和功能过程中，介导根尖蛋白靶向和根尖管腔协调形成的分子机制。最近的工作，包括我们之前的资助发表的工作，发现Rab11 GTPase是上皮细胞极性和上皮组织形态发生的关键调节因子。与所有rabb gtpase一样，Rab11通过结合和激活各种效应蛋白发挥作用。因此，破译这些效应蛋白的作用是理解上皮细胞功能的关键一步。在过去的十年中，已经鉴定了几种Rab11结合蛋白，其中包括Rab11家族相互作用蛋白，也称为FIPs。包括我们在内的几个实验室的研究表明，FIP家族的FIP5成员调节上皮细胞极化蛋白的转运和根尖管腔的形成。此外，我们已经证明FIP5通过结合并激活分选连接蛋白18、激酶蛋白II和环粘蛋白作为支架因子。最后，我们发现FIP5/cingulin复合物在末期后期被招募到中间体，这一事件在建立单个新生顶管中起着关键作用。根据最近发表的结果和我们的初步数据，我们提出以下假设。首先，在根尖管腔形成过程中，FIP5/cingulin复合物介导根尖内体靶向。其次，在终末期晚期，FIP5/cingulin在中体周围靶向是根尖管腔形成所必需的。因此，本提案旨在在体外（MDCK细胞）和体内（斑马鱼）模型中测试这些假设。这项建议有三个具体目标。在目的1中，我们将分析FIP5和环蛋白相互作用在根尖管腔形成过程中调节根尖蛋白运输中的作用。在目的#2中，我们将测试中间体和细胞分裂对顶管起始的作用和调节。最后，在目的#3中，我们将使用斑马鱼肾原管和肠道形成模型来测试FIP5/cingulin依赖的内吞运输在上皮组织形态发生中的作用。该项目的完成将为理解上皮组织形态发生和重塑过程中上皮细胞极化和根尖管腔形成的分子机制和调控提供新的见解。

DESCRIPTION (provided by applicant): Kidney and gut epithelial cells are structurally and functionally polarized to transport specific molecules while maintaining a trans-epithelial barrier This cellular asymmetry is essential for the proper functioning of epithelial tissues and depends on polarized endocytic transport routes. Additionally, epithelial cells coordinate their polarizatin with neighboring cells to form an apical lumen, a key step in the establishment of renal and gut architecture, and thereby function. Indeed, malfunctions in polarized epithelial transport and apical lumen formation are responsible for a variety of renal and intestinal disorders. This proposal aims to fill critical gaps in our understanding of epithelial cell biology, which are the molecular mechanisms mediating apical protein targeting and coordinated formation of apical lumen during kidney morphogenesis and function. Recent work, including work published from our preceding grant, identified Rab11 GTPase as a key regulator of epithelial cell polarity and epithelial tissue morphogenesis. As all Rab GTPases, Rab11 functions by binding and activation various effector proteins. Thus, deciphering the roles of these effector proteins is a key step in understanding the function of epithelial cell. During the last decade, several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Work from several laboratories, including ours, has shown that FIP5 member of FIP family regulates polarized protein transport and apical lumen formation in epithelial cells. Furthermore, we have shown that FIP5 acts as a scaffolding factor by binding to and activating sorting nexin 18, kinesin II and cingulin. Finally, we have shown that FIP5/cingulin complex is recruited to the midbody during late telophase, the event that plays a key role in establishing a single nascent apical lumen. Based on recently published results and on our preliminary data, we propose the following hypotheses. First, FIP5/cingulin complex mediates apical endosome targeting during apical lumen formation. Second, FIP5/cingulin targeting around the midbody during late telophase is required for apical lumen formation. Thus, this proposal is designed to test these hypotheses in both in vitro (MDCK cells) and in vivo (zebrafish) models. This proposal has three specific aims. In the aim #1 we will analyze the role of FIP5 and cingulin interaction in regulating apical protein transport during apical lumen formation. In the aim #2 we will test the role and regulation of the midbody and cytokinesis for the initiation of the apical lumen. Finally, in aim #3 we will test the role of FIP5/cingulin-dependent endocytic transport during epithelial tissue morphogenesis using zebrafish pronephric tube and intestinal tract formation models. Completion of this project will provide a novel insight in understanding the molecular machinery and regulation of epithelial cell polarization and apical lumen formation during epithelial tissue morphogenesis and remodeling.