Mechanisms of Primary Cilium Assembly and Disassembly

项目摘要 初级纤毛是一个微米级的结构，突出于人体大多数细胞的表面 身体纤毛曾经被认为是退化的，最近被证明在胚胎发育中起关键作用。 发育、感官知觉和组织稳态。纤毛的两个关键功能导致了这些 生理作用：纤毛是多种信号通路和结构的组织中心， 分解与细胞周期的进程紧密相连。与这些作用一致，睫状体缺陷 引起小儿疾病，称为纤毛病，并可促进肿瘤发生。最近的这些发现 强调了纤毛的重要性，但也强调了我们知识中的许多空白。关键问题包括： 纤毛是如何组装、维持和分解的，蛋白质是如何进出纤毛的， 促进信号传导，以及纤毛解体如何与细胞周期进程相关联？目前，许多基因 支持纤毛功能的产品尚未被详细鉴定或表征，因此， 这些问题仍然难以捉摸。 我的实验室旨在通过结合细胞- 我们已经开发了新的方法，包括基于CRISPR的功能筛选， 在半透化细胞中的体外重建。特别是，我们最近进行了一次全基因组筛查， 通过Hedgehog（Hh）途径确定纤毛依赖性信号传导所需的基因。此屏幕 以高精度和灵敏度鉴定了命中基因，揭示了纤毛组装和Hh所需的新基因， 信号，并提出了纤毛和疾病之间的新联系。我们现在建议在这个屏幕上建立 通过1）功能性地表征新鉴定的命中基因，包括我们发现需要的Rab GT3 用于纤毛发生并定位于纤毛，以及2）调整我们的CRISPR筛选工具，以系统地研究 纤毛功能的一个方面仍然知之甚少：初级纤毛的调节分解。我们的工作 的纤毛拆卸将集中在假设纤毛拆卸监测在一个检查点样 在不受控制的细胞生长的情况下，例如在肿瘤发生期间，可能失调。此外 为了进行遗传筛选以确定纤毛解体的介质和调节剂，我们将解剖 通过互补的活体成像分析和体外重建的拆卸机制。这些后者 实验将利用我开发的半透化细胞系统， 纤毛过程的生化分析，包括纤毛解体。 总之，这个项目的目的是提供基本的见解初级纤毛，将扩大我们的研究。 了解细胞周期，蛋白质运输，信号转导和细胞器生物发生。此外，本发明还 这些研究将有助于揭示纤毛缺陷如何导致纤毛病和肿瘤发生。

PROJECT SUMMARY The primary cilium is a micron-scale structure that protrudes from the surface of most cells in the human body. Once thought to be vestigial, the cilium has recently been shown to have key roles in embryonic development, sensory perception, and tissue homeostasis. Two key functions of cilia give rise to these physiologic roles: cilia are both organizing centers for diverse signaling pathways and structures whose assembly and disassembly is tightly linked to progression through the cell cycle. Consistent with these roles, ciliary defects cause pediatric disorders known as ciliopathies and can promote tumorigenesis. These recent discoveries have highlighted the importance of cilia but also underscored many gaps in our knowledge. Key questions include: how are cilia assembled, maintained, and disassembled, how do proteins traffic to and from cilia, how do cilia promote signaling, and how is cilium disassembly linked to cell cycle progression? At present, many gene products that support cilium function have yet to be identified or characterized in detail, and thus the answers to these questions remain elusive. My lab aims to understand the molecular basis of mammalian primary cilium function by combining cell- based assays with new approaches we have developed including CRISPR-based functional screening and in vitro reconstitution in semi-permeabilized cells. In particular, we recently conducted a genome-wide screen to identify genes required for cilium-dependent signaling through the Hedgehog (Hh) pathway. This screen identified hit genes with high precision and sensitivity, revealed new genes required for cilium assembly and Hh signaling, and suggested new connections between cilia and disease. We now propose to build on this screen by 1) functionally characterizing newly identified hit genes, including a Rab GTPase that we find to be required for ciliogenesis and to localize to cilia, and 2) adapting our CRISPR screening tools to systematically investigate an aspect of cilium function that remains poorly understood: the regulated disassembly of primary cilia. Our work on cilium disassembly will focus on the hypothesis that cilium disassembly is monitored in a checkpoint-like manner and may be dysregulated in cases of uncontrolled cell growth, such as during tumorigenesis. In addition to conducting a genetic screen to identify mediators and regulators of cilium disassembly, we will dissect the mechanism of disassembly through complementary live-imaging assays and in vitro reconstitution. These latter experiments will take advantage of a semi-permeabilized cell system I developed that allows powerful biochemical analysis of ciliary processes, including cilium disassembly. Taken together, this project aims to provide fundamental insights into primary cilia that will broaden our understanding of the cell cycle, protein trafficking, signal transduction, and organelle biogenesis. Additionally, these studies will help to reveal how ciliary defects contribute to ciliopathies and tumorigenesis.