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

描述(由申请人提供)：细胞功能严重依赖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转运媒介的失调有关。外囊是这些关键的转运媒介之一，它是一种从酵母到哺乳动物的八聚体细胞质复合体[1]。外囊最为人所知的是它作为一种连接复合体的角色，参与在广泛的关键细胞过程中在质膜上进行定向囊泡融合。然而，关于外囊是如何调节的，以及它在细胞中的功能是什么，仍有许多未知之处。越来越清楚的是，外囊并不总是作为一个单一的单位发挥作用；相反，不同的亚单位分别与不同的外囊介导的过程有关。我们的实验室最近公布了在秀丽线虫体内进行的全基因组RNAi筛查，该筛查强烈地暗示外囊的亚单位是细胞程序的重要组成部分，该程序在受到造孔毒素(PFT)蛋白的攻击后对质膜上形成的孔做出反应[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]。对PFT的研究不仅对于了解细菌的发病机制很重要，而且对于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)途径的信号传递以及膜运输事件，包括增加内吞作用、质膜脱落和膜重新密封[2，6，7]。同样的反应也在哺乳动物细胞中观察到[8-12]。由于外囊还与MAPK信号转导通路[13-15]以及在其他环境中的膜运输[16]有关，我的目的是利用线虫肠道中PFT诱导的反应的研究作为探索外囊功能和调节的新系统。我将使用RNAi来确定与细胞对PFTs的反应相关的信号转导和膜运输事件所需要的外囊亚基。我还将构建一种表达荧光标记的胞外囊亚单位的报告虫体，以观察胞囊激活，并随后确定先前发现的哪些PFT防御基因[2]调节胞囊激活。这些研究结果将有助于阐明胞囊及其组成亚单位在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.