Project 1: Regulation of polarized exocytosis during epithelial differentiation

项目摘要/摘要--项目1 真核生物中一个重要的细胞生理调节因子是八蛋白胞囊复合体，它 运输胞内小泡的亚群以进行胞吐到质膜上的特定位置。近期 对哺乳动物上皮细胞的研究表明，外囊在几个方面的分化中都是至关重要的 和形态发生，如上皮屏障的完整性，囊和管腔的形成，以及组装和 初级纤毛的信号。然而，仍然知之甚少的是其分子机制 上皮细胞引导外囊完成许多不同的功能。识别和表征 这些机制将是更好地理解哺乳动物的发育和生理的核心。 上皮组织。我们最近建立了新的模型系统来研究外囊的调节。 细胞转运和组织发育，包括第一次组织特异性胞囊条件性敲除 老鼠模型。来自我们对肾脏发育过程中的胞囊的研究，并与一个研究所合作 生物发生研究Cobre-1项目负责人，我们发现了有趣的相似之处和差异 肾囊化和植入前囊泡空化过程中的外囊调节。基于我们的 初步发现，我们的假设是，在分化的每个阶段，上皮细胞都显示出一系列 调控机制的时间和空间控制外囊介导的转运，这是至关重要的正常 上皮组织形态发生。我们将通过以下具体目标来检验这一假设：(1)确定 上皮特异的调控机制，重定向胞外包囊介导的运输在间充质到- 上皮转化(MET)。在这里，我们使用MET的诱导多能(IPS)细胞模型和MET的体内模型。 在胚泡形成和肾脏发育期间使用MET，以评估对可能 诱导上皮性货物和运输的专一性。(2)确定外囊是否引导极化的 单向液体转运所必需的溶质载体和水通道的胞吐作用 上皮腔。我们将使用我们的新的排囊条件基因敲除小鼠，并从 这些小鼠，测试外囊是否介导了关键的溶质转运体和水通道的转位 在囊胚发生过程中，比较囊泡空化和肾泡形成的差异。(3) 确定在极化上皮细胞中控制胞囊向初级纤毛运输的生化相互作用 细胞及其对胚泡形态发生的重要性。我们将调查八个外囊中的哪一个 亚基定位于纤毛，测试纤毛发生所必需的物质，并筛选外囊结合蛋白。 尤其是纤毛。我们还将研究囊外囊在胚泡内初级纤毛运输中的作用。 该项目的预期结果是表征新的遗传和生化机制， 哺乳动物上皮组织用来控制分化、生长和分化过程中的极化胞吐。 形态发生。

PROJECT SUMMARY/ABSTRACT - Project 1 An important regulator of cell physiology in eukaryotes is the eight-protein exocyst complex, which traffics subsets of intracellular vesicles for exocytosis to particular sites on the plasma membrane. Recent studies of mammalian epithelial cells have shown the exocyst is critical for several aspects of differentiation and morphogenesis, such as epithelial barrier integrity, cyst and tubule lumen formation, and assembly and signaling of primary cilia. However, what remain poorly understood are the molecular mechanisms by which epithelial cells direct the exocyst to accomplish so many different functions. Identifying and characterizing these mechanisms will be central to better understanding the development and physiology of mammalian epithelial tissues. We have recently established novel model systems to investigate the exocyst's regulation of cellular trafficking and tissue development, including the first tissue-specific exocyst conditional knockout mouse model. From our studies of the exocyst during renal development, and in collaboration with an Institute of Biogenesis Research COBRE-1 Project Leader, we have identified interesting parallels and variances of exocyst regulation during renal cystogenesis and pre-implantation blastocyst cavitation. Based on our preliminary findings, our hypothesis is that epithelial cells, at each stage of differentiation, display an array of regulatory mechanisms to temporally and spatially control exocyst-mediated trafficking, which is vital to normal epithelial tissue morphogenesis. We will test this hypothesis through the following Specific Aims: (1) Identify epithelial-specific regulatory mechanisms that redirect exocyst-mediated trafficking during mesenchymal-to- epithelial transition (MET). Here we use an induced pluripotent (iPS) cell model of MET, and in vivo models of MET during blastocyst formation and kidney development, to evaluate modifications of the exocyst that may induce the specificity of epithelial cargo and trafficking. (2) Determine if the exocyst directs the polarized exocytosis of solute carriers and aquaporin channels necessary for unidirectional fluid transport into growing epithelial lumens. We will use our novel exocyst conditional knockout mice, and cell lines established from these mice, to test if the exocyst mediates translocation of key solute transporters and aquaporin channels during cystogenesis, comparing differences during blastocyst cavitation and renal vesicle formation. (3) Identify the biochemical interactions that control the exocyst's trafficking to primary cilia in polarized epithelial cells and their importance to blastocyst morphogenesis. We will investigate which of the eight exocyst subunits localize to the cilia, test which are necessary for ciliogenesis, and screen for exocyst-binding proteins specifically in the cilia. We will also investigate the exocyst's role in primary cilia trafficking in the blastocyst. The anticipated outcome of the project is characterization of new genetic and biochemical mechanisms that mammalian epithelial tissues use to control polarized exocytosis during their differentiation, growth, and morphogenesis.