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至其正确目的地的能力，并且诸如癌症和多囊肾疾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外囊，一种从酵母到哺乳动物保守的八聚体胞质复合物[1]。外囊最为人所知的是它作为一个拴系复合物的作用，在广泛的关键细胞过程中参与质膜上的定向囊泡融合。然而，关于外囊是如何调节的以及它在细胞中的功能仍然是未知的。越来越清楚的是，外囊并不总是作为一个单一的单位;相反，不同的亚基已经单独连接到不同的外囊介导的过程。我们的实验室最近发表了在秀丽隐杆线虫蛔虫中进行的全基因组RNAi筛选，其强烈暗示外囊的亚基是细胞程序的重要组成部分，该细胞程序响应于在孔形成毒素（PFT）蛋白攻击后在质膜中形成的孔[2]。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、炭疽杆菌和霍乱弧菌[3-5]。PFT的研究不仅对于理解细菌的发病机制很重要，而且对于理解细胞一般如何处理膜损伤也很重要。我们的实验室已经发现C.秀丽线虫的细胞内信号转导包括通过丝裂原活化蛋白激酶（MAPK）途径的信号转导以及膜运输事件，包括增加的内吞作用、质膜脱落和膜重新密封[2，6，7]。在哺乳动物细胞中也观察到了这些相同的反应[8-12]。由于外囊也与MAPK信号转导途径[13-15]和其他情况下的膜运输[16]有关，因此我的目的是使用PFT诱导的C.线虫肠作为一个新的系统，探索外囊的功能和调节。我将使用RNAi来确定哪些外囊亚基是与细胞对PFT的反应相关的信号转导和膜运输事件所必需的。我还将构建一个报告蠕虫表达荧光标记的外囊亚基，以观察外囊激活，并随后确定哪些先前鉴定的PFT防御基因[2]调节外囊激活。这些研究的结果将揭示新的光的作用和调控的外囊及其组成亚基在基本的膜运输过程中，并将有重要的意义，了解细胞如何处理膜损伤和控制细菌感染和控制疾病的发病机制涉及失调的外囊运输。

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.