The exocyst in ciliogenesis and cystogenesis

描述(由申请人提供)： 我们的目标是了解高度保守的八蛋白胞囊复合体在纤毛发生和囊变中的作用。包囊是肾脏等上皮性器官的“积木”，包囊形成缺陷与人类疾病有关，如常染色体显性遗传性多囊肾病(ADPKD)。ADPKD是最常见的潜在致命性遗传病，影响约1/500人，是终末期肾病的第四大原因。目前，包括10万名退伍军人和家属在内的50万美国ADPKD患者还没有获得批准的治疗方法。在肾小管细胞中，我们最近发现外囊复合体定位于初级纤毛的形成，并且对于初级纤毛的形成是必要的，初级纤毛是与ADPKD发病密切相关的细胞器。胞囊与中心纤毛蛋白Par3共定位和免疫共沉淀。在敲除外囊成分Sec10后，不再发生原发纤毛发生。我们的初步研究还表明，Sec10和Par3之间存在相互作用，这是Aim 1的基础。分泌途径对细胞的正常功能至关重要，而外囊则介导携带膜蛋白的分泌囊泡的靶向和对接。多种Rho和Rab家族的GTP酶调节外囊。在酵母中，我们发现Rho GTP酶CDC42定位于特定的质膜结构域，我们的初步研究表明：Sec10和CDC42是结合伙伴，CDC42和Sec10共同定位于初生纤毛，而CDC42的扰动抑制了纤毛的发生。我们还发现，胞囊与Rab8免疫共沉淀，Rab8是纤毛发生所必需的一种小GTP酶，发现于携带纤毛蛋白的囊泡表面。这些都是AIM 2的关键数据。我们的初步研究表明，Sec10基因敲除细胞在几个关键方面与ADPKD细胞相似：它们的细胞内钙水平较低，不会随着液体流动而增加，丝裂原活化蛋白激酶(MAPK)途径被启动，并且它们具有高增殖能力。重要的是，我们证明了外囊Sec10和多囊蛋白-2之间的生化相互作用。编码钙通道多囊蛋白-2的PKD2是两个基因之一，当突变时，会导致ADPKD。我们使用sec10反义吗啉在斑马鱼体内显示了相关性，这导致了与注射PKD2吗啉后非常相似的表型，尾巴卷曲，肾小球扩张，纤毛紊乱，MAPK通路激活。最后，我们证明了PKD2和Sec10之间的遗传相互作用，在每个吗啡单独没有影响的少量，共同导致上述表型。我们的合作者发现，胞外包囊错误定位于人ADPKD细胞内，从而建立了胞外包囊定位与ADPKD之间的联系。这些数据是目标3的基础。因此，我们提出的研究针对的假设是，外囊在纤毛发生和囊变中的中心作用是通过CDC42将其定位在初级纤毛上，通过与Par3结合来稳定它，以及随后靶向和对接携带纤毛蛋白的Rab8阳性囊泡来实现的。因此，我们将根据我们的发现，通过实现三个目标来确定外囊对于纤毛发生是必要的。在目标1中，我们将识别和定位胞囊成分Sec10和Par3之间的相互作用区域。然后，我们将破坏相互作用的结构域，并确定与纤毛发生和囊变有关的后果。在目标2中，我们将研究Rho和Rab GTP酶，特别是CDC42和Rab8，在定位外囊到初级纤毛以运输携带纤毛蛋白的分泌囊泡中的作用。最后，在目标3中，我们将利用斑马鱼Sec10和Rho/Rab GTP酶的反义Morolinos来确定外囊在体内纤毛发生和囊变中的作用。这些目标的成功完成将提高我们在细胞和分子水平上对蛋白质递送、纤毛发生和囊变的理解，并为ADPKD的治疗干预找到新的靶点。 公共卫生相关性： 常染色体显性遗传性多囊肾病(ADPKD)是最常见的潜在致命性遗传病，是肾衰竭的第四大原因，影响约1/500人，即500,000美国人。考虑到2500万退伍军人和另外4500万家庭成员，至少有10万退伍军人或家属患有ADPKD。作为费城VAMC的肾内科主任和宾夕法尼亚州陆军国民警卫队的内科医生，我经常看到ADPKD患者，很难告诉他们没有批准的治疗方法。ADPKD的特点是囊性过度生长，破坏肾脏。一种被称为初级纤毛的细胞器与ADPKD的发病密切相关，我们的初步研究表明，在细胞培养和活体动物中，8蛋白外囊复合体是纤毛和包囊形成所必需的。在这里，我们建议进行实验，以测试外囊如何建立初级纤毛，并成功完成，从而识别出干预ADPKD的新的候选靶点。

DESCRIPTION (provided by applicant): Our goal here is to understand the role of the highly conserved eight-protein exocyst complex in ciliogenesis and cystogenesis. Cysts are "building blocks" for epithelial organs such as the kidney, and defects in cyst formation are implicated in human disorders such as autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most common potentially lethal genetic disease, affecting approximately 1/500 people, and the fourth leading cause of end-stage kidney disease. There are currently no approved therapies for the 500,000 Americans, including 100,000 veterans and dependents, with ADPKD. In renal tubule cells, we recently showed that the exocyst complex localized to, and was necessary for, formation of the primary cilium, an organelle strongly implicated in ADPKD pathogenesis. The exocyst co- localized and co-immunoprecipitated with Par3, a central ciliary protein. Following knockdown of exocyst component Sec10, primary ciliogenesis no longer occurred. Our Preliminary Studies also show an interaction between Sec10 and Par3, which is the foundation for Aim 1. The secretory pathway is crucial for proper cell function and the exocyst is known for mediating the targeting and docking of secretory vesicles carrying membrane proteins. Multiple Rho and Rab family GTPases regulate the exocyst. In yeast, we showed that Cdc42, a Rho GTPase, localized the exocyst to specific plasma membrane domains and our Preliminary Studies demonstrate that: Sec10 and Cdc42 are binding partners, Cdc42 and Sec10 co-localize at primary cilia, and Cdc42 perturbation inhibits ciliogenesis. We also show that the exocyst co-immunoprecipitates with Rab8, a small GTPase necessary for ciliogenesis and found on the surface of vesicles carrying ciliary proteins. These are critical data for Aim 2. Our Preliminary Studies show that Sec10 knockdown cells are similar to ADPKD cells in several crucial ways: they have low levels of intracellular calcium that do not increase with fluid flow, the mitogen activated protein kinase (MAPK) pathway is turned on, and they are hyperproliferative. Importantly, we demonstrate a biochemical interaction between exocyst Sec10 and polycystin-2. PKD2, encoding the calcium channel polycystin-2, is one of two genes, which when mutated, causes ADPKD. We show in vivo correlation using sec10 antisense morpholinos in zebrafish, which results in a remarkably similar phenotype to that following injection of pkd2 morpholinos, with a "curly up" tail, dilated glomeruli, disorganized cilia, and MAPK pathway activation. Finally, we demonstrate a genetic interaction between pkd2 and sec10, in that small amounts of each morpholino that individually have no effect, together result in the phenotype noted above. Our collaborators showed the exocyst mislocalized intracellularly in human ADPKD cells, thereby establishing a link between exocyst localization and ADPKD. These data are the basis for Aim 3. Our proposed research, therefore, is directed toward the hypothesis that the central role the exocyst plays in ciliogenesis and cystogenesis is mediated via its localization at the primary cilium by Cdc42, its stabilization by binding to Par3, and the subsequent targeting and docking of Rab8 positive vesicles carrying ciliary proteins. Accordingly, we will build on our findings that the exocyst is necessary for ciliogenesis by performing three Aims. In Aim 1, we will identify and map the interacting domain between exocyst component Sec10 and Par3. We will then disrupt the interacting domain and determine the consequences with respect to ciliogenesis and cystogenesis. In Aim 2, we will investigate the role of Rho and Rab GTPases, especially Cdc42 and Rab8, in localizing the exocyst to primary cilia for trafficking of secretory vesicles carrying ciliary proteins. Finally, in Aim 3, we will determine the role of the exocyst in ciliogenesis and cystogenesis in vivo using antisense morpholinos of sec10 and Rho/Rab GTPases in zebrafish. Successful completion of these Aims will improve our understanding of protein delivery, ciliogenesis, and cystogenesis at the cell and molecular levels, and identify novel targets for therapeutic intervention in ADPKD. PUBLIC HEALTH RELEVANCE: Autosomal dominant polycystic kidney disease (ADPKD), affecting approximately 1/500 people, or 500,000 Americans, is the most common potentially lethal genetic disease and 4th leading cause of kidney failure. Given 25 million veterans and another 45 million family members, there are at least 100,000 veterans or dependents with ADPKD. As Chief of Kidney Medicine at the Philadelphia VAMC and physician in the Pennsylvania Army National Guard, I frequently see patients with ADPKD, and it is difficult to tell them that there are no approved therapies. ADPKD is characterized by cystic overgrowth that destroys the kidney. A cellular organelle called the primary cilium has been strongly implicated in ADPKD pathogenesis and our Preliminary Studies show that the eight-protein exocyst complex is necessary for cilia and cyst formation, both in cell culture and living animals. Here, we propose experiments to test how the exocyst builds primary cilia, with successful completion leading to identification of novel candidate targets for intervention in ADPKD.