The role of a cilium assembly subnetwork at the basal body-vesicle membrane interface

初级纤毛是延伸到细胞外的细胞触角，调节关键的信号通路。纤毛由根植于基底的微管轴组成，周围环绕着一层特殊的膜，膜上集中着受体，以正确解释不同的细胞外信号。纤毛缺陷会导致多种疾病。这些疾病包括多囊肾病、视网膜变性和先天性心脏缺陷，这些细胞器的丢失可能是癌症进展过程中的一个关键事件。我研究的长期目标是了解睫状膜和潜在结构是如何组装和维护的。虽然这个细胞器的许多成分已经被鉴定出来，但这些蛋白质是如何相互作用的，或者是如何与细胞环境相互作用发挥其功能的还不清楚。目前的提议旨在了解纤毛形成过程中如何涉及一种新的蛋白质网络-COMMD/WASH-从膜上分拣货物。我们在一项大型蛋白质组学研究中发现了这个复合体，我们有证据表明，它与已知与新生睫状膜相关的基底体成分有关。我们也有数据表明，细胞产生正常纤毛是必需的。我们将利用基因中断、超分辨显微镜和蛋白质组学来研究：1)这个复合体的哪些成分是起作用所必需的；2)这些组分具体影响起作用的步骤；3)这个复合体的货物分拣功能如何影响纤毛膜。*我们将利用这一知识在分子水平上了解纤毛形成过程中基底体的蛋白质如何与膜结合，从而产生功能正常的睫状膜。在这样做的过程中，我们将提供基本的见解来帮助理解，不仅是细胞生长和信号传递，还包括如何更好地治疗人类疾病。拟议的研究计划将培训三名研究生和多名本科生，学习基础细胞和分子遗传学方法，以及蛋白质组学和功能基因组学的尖端新方法。这种培训将推动下一代加拿大科学家的职业生涯。出于这些原因，这项研究对加拿大的健康和知识经济以及整个加拿大人都有长期的好处。*****

Primary cilia are cellular antennae' that extend out of the cell and mediate critical signaling pathways. A cilium consists of a microtubular shaft which is rooted at the basal body, and surrounded by a specialized membrane that is concentrated with receptors to correctly interpret diverse extracellular cues. Ciliary defects lead to large spectrum of diseases. These include polycystic kidney disease, retinal degeneration and congenital heart defects, and the loss of this organelle may be a key event during cancer progression.******The long term objective of my research is to understand how the ciliary membrane and underlying structures are assembled and maintained. While many of the constituents of this organelle have been identified, it is unclear how these proteins interact with each other or with the cellular milieu for their function. The current proposal is designed to understand how a novel network of proteins - COMMD/WASH - that sort cargo from membranes is involved during cilium formation. We found this complex in a large proteomic study, and we have evidence that it associates with components of the basal body known to associate with the nascent ciliary membrane. We also have data indicating that it is required for cells to produce normal cilia. We will use gene disruption, super-resolution microscopy and proteomics to study : 1) which components of this complex are required for ciliation; 2) which exact step of ciliation is affected by these components; 3) how the cargo sorting function of this complex affects the ciliary membrane.******We will leverage this knowledge to understand, at a molecular level, how the proteins at the basal body interface with membranes during cilium formation, so as to produce a functioning ciliary membrane. In doing so, we will provide fundamental insights to help understand, not only cellular growth and signalling, but also how to better treat human disease. The proposed research program will train three graduate students and multiple undergraduate associates in fundamental cell and molecular genetic methods, as well as in cutting-edge new approaches in proteomics and functional genomics. This training will propel the next generation of Canadian scientists in their careers. For these reasons, this research has long-term benefits to Canada's health and knowledge economies, as well as to Canadians at large. *****