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Mechanisms of Primary Cilium Assembly and Disassembly

初级纤毛组装和拆卸的机制

基本信息

批准号：
10027412
负责人：
David King Breslow
金额：
$ 40.53万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10027412
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

项目摘要

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