Reconstructing Primary Cilia

描述(由申请人提供) 摘要：初级纤毛是一种微米级的信号机器，从我们体内的大多数细胞投射出来，是检测和处理光、化学和机械信号所必需的。出生缺陷、肥胖、智力低下和囊性器官只是纤毛受损患者的疾病表型的一部分。虽然在过去的十年里已经编制了一个由数百种蛋白质组成的纤毛的广泛部件清单，但未来的一个主要挑战是了解这些蛋白质如何协同工作，以允许这种非凡结构的自组装和功能。这一建议是基于这样的想法，即在我们对初级纤毛的理解方面的重大进展将源于使用无细胞提取物在体外重建这种细胞器的组装和功能的系统努力。这种生化分析将利用与人类疾病有关的纤毛蛋白的定量遗传相互作用分析。我提出了这一方法的蓝图，整合了传统的蛋白质生物化学、现代成像和组合RNAi，以了解中心体、膜和可溶性因子是如何协作形成功能纤毛的。这种重组将允许对纤毛疾病基因编码的蛋白质进行高分辨率分析，并最终开发出修复甚至重新设计纤毛功能的生化策略。 与公共卫生相关：我们体内的大多数细胞通过一种名为初级纤毛的天线状结构与环境进行交流。纤毛受损可导致人类多种疾病，包括出生缺陷、肥胖、智力低下、失明和肾脏异常。通过了解这个重要的细胞通讯中心信号是如何处理的，我们希望了解纤毛相关的疾病，并开发修复纤毛功能的新策略。

DESCRIPTION (Provided by the applicant) Abstract: The primary cilium is a micron-scale signaling machine that projects from most cells in our bodies and is required for the detection and processing of optical, chemical and mechanical signals. Birth defects, obesity, mental retardation, and cystic organs are just a subset of the disease phenotypes seen in patients with damaged cilia. While an extensive parts list for cilia consisting of hundreds of proteins has been compiled over the last decade, a major challenge going forward is to understand how these proteins work together to allow the self-assembly and function of this remarkable structure. This proposal is based on the idea that major advances in our understanding of primary cilia will result from a systematic effort to reconstitute the assembly and function of this organelle in vitro using cell-free extracts. This biochemical analysis will draw on quantitative genetic interaction analysis of cilia proteins implicated in human diseases. I present a blueprint for this approach, integrating traditional protein biochemistry, modern imaging, and combinatorial RNAi, to understanding how centrosomes, membranes, and soluble factors cooperate to form functional cilia. This reconstitution will allow a high-resolution analysis of proteins encoded by cilia disease genes and, ultimately, the development of biochemical strategies to repair and even re-engineer ciliary function. Public Health Relevance: Most cells in our bodies communicate with the environment through an antenna-like structure called the primary cilium. Damage to cilia can cause a variety of diseases in humans, including birth defects, obesity, mental retardation, blindness, and kidney abnormalities. By understanding how signals are processed in this important cellular communication center, we hope to understand cilia-related diseases and develop new strategies to repair cilia function.