Project 1: Regulation of polarized exocytosis during epithelial differentiation

项目总结/摘要-项目1 真核生物中细胞生理学的一个重要调节因子是八蛋白外囊复合物， 将胞内小泡的亚群转运到质膜上的特定位点进行胞吐作用。最近 对哺乳动物上皮细胞的研究表明，外囊对于分化的几个方面是至关重要的 和形态发生，如上皮屏障完整性，囊肿和小管腔形成，以及组装和 初级纤毛的信号。然而，人们对这种现象的分子机制仍然知之甚少 上皮细胞引导外囊完成许多不同的功能。鉴定和表征 这些机制将是更好地理解哺乳动物的发育和生理学的核心。 上皮组织我们最近建立了新的模型系统，以研究外囊的调节， 细胞运输和组织发育，包括第一个组织特异性外囊条件性敲除 小鼠模型根据我们对肾脏发育过程中的外囊的研究，并与一个研究所合作， 生物发生研究COBRE-1项目负责人，我们已经确定了有趣的相似之处和差异， 肾囊肿发生和植入前胚泡空化过程中的外囊调节。基于我们 初步发现，我们的假设是，上皮细胞，在每个阶段的分化，显示一系列的 调节机制，以在时间和空间上控制外囊介导的运输，这对正常 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）识别 上皮特异性调节机制，在间充质细胞到上皮细胞的过程中重定向外来囊介导的运输， 上皮转化（MET）。在这里，我们使用MET的诱导多能（iPS）细胞模型，以及MET的体内模型。 MET在胚泡形成和肾脏发育过程中的作用，以评估可能 诱导上皮货物和运输的特异性。(2)确定外囊是否引导极化的 溶质载体和水通道蛋白通道的胞吐作用是单向流体运输到生长中所必需的 上皮腔我们将使用我们的新的外囊条件性敲除小鼠，和细胞系建立从 这些小鼠，以测试外囊是否介导关键溶质转运蛋白和水通道蛋白通道的易位 在囊形成过程中，比较胚泡空化和肾泡形成过程中的差异。（三） 确定控制外囊向极化上皮细胞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亚基，并筛选外泌囊结合蛋白 特别是纤毛我们也将调查外囊的作用，初级纤毛贩运的囊胚。 该项目的预期成果是表征新的遗传和生化机制， 哺乳动物上皮组织在其分化、生长和分化期间用于控制极化胞吐作用， 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.