Cellular responses to pore-forming toxins and regulation/function of the exocyst

描述（由申请人提供）：细胞功能主要取决于细胞将蛋白质和膜引导到正确目的地的能力，癌症和多囊肾病等疾病与重要运输介质的失调有关。这些关键的运输介质之一是胞囊，这是一种从酵母到哺乳动物的八聚体细胞质复合体。胞囊最为人所知的是其作为一种系栓复合物的作用，在广泛的关键细胞过程中参与了质膜上的定向囊泡融合。然而，关于胞囊是如何被调节的，以及它在细胞中的功能是什么，我们仍然知之甚少。也越来越清楚的是，囊泡并不总是作为一个单一的单位；相反，不同的亚基单独与不同的囊泡介导过程相关联。我们的实验室最近发表了一项全基因组RNAi筛选，该筛选在秀丽隐杆线虫中进行，强烈暗示胞囊亚基是细胞程序的重要组成部分，这些程序是在形成孔毒素（PFT）蛋白[2]攻击后对质膜上形成的孔作出反应的。pft是最常见的细菌毒力因子，由几种主要的人类病原体分泌，包括金黄色葡萄球菌、炭疽芽孢杆菌和霍乱弧菌[3-5]。PFTs的研究不仅对了解细菌发病机制很重要，而且对了解细胞如何处理一般的膜损伤也很重要。我们的实验室已经发现秀丽隐杆线虫的PFT防御包括通过丝裂原活化蛋白激酶（MAPK）途径的信号传导以及膜运输事件，包括增加的内吞作用、质膜脱落和膜重封[2,6,7]。在哺乳动物细胞中也观察到同样的反应[8-12]。由于外囊也与MAPK信号转导通路[13-15]和其他情况下的膜运输有关[13-15]，我的目的是利用研究秀丽隐杆线虫肠道中pft诱导的反应作为探索外囊功能和调控的新系统。我将使用RNAi来确定哪些胞囊亚基是与细胞对pft反应相关的信号转导和膜运输事件所必需的。我还将构建表达荧光标记的囊泡亚基的报告虫，以观察囊泡激活，并随后确定哪些先前鉴定的PFT防御基因[2]调节囊泡激活。这些研究结果将揭示胞囊及其组成亚基在基本膜运输过程中的作用和调控，并将对理解细胞如何处理膜损伤、控制细菌感染和控制发病机制涉及胞囊运输失调的疾病具有重要意义。

DESCRIPTION (provided by applicant): Cellular function depends critically on the ability of the cell to direct proteins and membranes to their correct destinations, and diseases such as cancer and polycystic kidney disease are associated with dysregulation of important trafficking mediators. One of these key trafficking mediators is the exocyst, an octameric cytoplasmic complex conserved from yeast to mammals [1]. The exocyst is best known for its proposed role as a tethering complex involved in directed vesicle fusion at the plasma membrane in a wide range of critical cellular processes. However, much is still unknown about how the exocyst is regulated and what its functions in the cell are. It is also becoming increasingly clear that the exocyst does not always act as a single unit; instead different subunits have been individually linked to different exocyst-mediated processes. Our lab recently published a genome-wide RNAi screen, performed in the Caenorhabditis elegans roundworm, that strongly implicates subunits of the exocyst as important components of the cellular program of responses to holes formed in the plasma membrane following attack by pore-forming toxin (PFT) proteins [2]. PFTs are the most common bacterial virulence factors and are secreted by several major human pathogens, including Staphylococcus aureus, Bacillus anthracis, and Vibrio cholerae [3-5]. Studies of PFTs are important not only for understanding bacterial pathogenesis but also for understanding how cells deal with membrane damage in general. Our lab has discovered that PFT defenses in C. elegans include signaling through mitogen activated protein kinase (MAPK) pathways as well as membrane trafficking events, including increased endocytosis, shedding of plasma membrane, and membrane resealing [2, 6, 7]. These same responses have also been observed in mammalian cells [8-12]. As the exocyst has also been connected to MAPK signal transduction pathways [13-15] and to membrane trafficking in other contexts [16], my aim is to use the study of PFT-induced responses in the C. elegans intestine as a new system for probing exocyst function and regulation. I will use RNAi to determine which exocyst subunits are required for the signal transduction and membrane trafficking events associated with cellular responses to PFTs. I will also construct a reporter worm expressing fluorescently-tagged exocyst subunits in order to observe exocyst activation and to subsequently determine which previously-identified PFT defense genes [2] regulate exocyst activation. Results from these studies will shed new light on the role and regulation of the exocyst and its component subunits in fundamental membrane trafficking processes and will have important implications for understanding how cells deal with membrane damage and for controlling bacterial infection and controlling of diseases whose pathogenesis involves dysregulation of exocytic trafficking.