Mechanisms of Primary Cilium Assembly and Disassembly

初级纤毛组装和拆卸的机制

• 批准号: 10430232
• 负责人: David King Breslow
• 金额: $ 41.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10430232
• 关键词: Biochemical; Biogenesis; Biological Assay; CRISPR screen; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cells; Childhood; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Defect; Development; Disease; Embryonic Development; Erinaceidae; Genes; Genetic Screening; Health; Homeostasis; Human body; Image; In Vitro; Knowledge; Link; Malignant Neoplasms; Mediator of activation protein; Molecular; Monitor; Organelles; Pathway interactions; Perception; Physiological; Proteins; Role; Screening procedure; Sensory; Signal Pathway; Signal Transduction; Structure; Structure of ciliary processes; Surface; Syndrome; System; Tissues; Work; base; cell growth; ciliopathy; cilium biogenesis; experimental study; gene product; genome wide screen; improved; insight; novel strategies; protein transport; rab GTP-Binding Proteins; reconstitution; screening; smoothened signaling pathway; tumor; tumorigenesis; uncontrolled cell growth

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.

项目总结 初级纤毛是一种微米级的结构，从人类大多数细胞的表面伸出。 尸体。纤毛曾经被认为是退化的，最近被证明在胚胎发育过程中起着关键作用。 发育、感觉知觉和组织动态平衡。纤毛的两个关键功能导致了这些 生理作用：纤毛既是不同信号通路的组织中心，也是其组装的结构 而拆卸与细胞周期的进展密切相关。与这些角色相一致的睫状肌缺陷 导致被称为纤毛病的儿科疾病，并可能促进肿瘤的发生。这些最近的发现已经 强调了纤毛的重要性，但也强调了我们知识中的许多差距。关键问题包括： 纤毛如何组装、维持和分解，蛋白质如何进出纤毛，纤毛如何 促进信号转导，纤毛分解如何与细胞周期进程相联系？目前，许多基因 支持纤毛功能的产品尚未被详细识别或表征，因此答案是 这些问题仍然难以捉摸。 我的实验室旨在通过将细胞- 基于我们开发的新方法的分析，包括基于CRISPR的功能筛选和 半透性细胞的体外重建。特别是，我们最近进行了全基因组筛查，以 确定纤毛依赖信号通过Hedgehog(HH)途径所需的基因。此屏幕 以高精度和高灵敏度鉴定HIT基因，发现纤毛组装和HH所需的新基因 信号，并提出纤毛和疾病之间的新联系。我们现在建议在此屏幕上构建 通过1)新发现的HIT基因的功能特征，包括我们发现需要的Rab GTP酶 用于纤毛发生和定位于纤毛，以及2)采用我们的CRISPR筛查工具系统地研究 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 纤毛拆解将集中于纤毛拆卸是在类似检查点的情况下受到监控的假设。 在细胞生长不受控制的情况下，例如在肿瘤形成过程中，可能会出现失控。此外 为了进行基因筛查以确定纤毛分解的介体和调节因子，我们将解剖 通过互补的活体成像分析和体外重建的拆解机制。这些是后者 实验将利用我开发的半渗透细胞系统，使强大的 纤毛过程的生化分析，包括纤毛分解。 综上所述，这个项目旨在提供对初级纤毛的基本见解，这将拓宽我们的 了解细胞周期、蛋白质运输、信号转导和细胞器生物发生。另外， 这些研究将有助于揭示纤毛缺陷是如何导致纤毛疾病和肿瘤发生的。