Mechanisms of Primary Cilium Assembly and Disassembly

Yale University, New Haven CT

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Abstract

PROJECT SUMMARY Cilia protrude from the surface of most cells in the human body and can be traced to the last eukaryotic common ancestor. Despite their near ubiquity, the functions of non-motile primary cilia were largely unknown until two foundational discoveries: first, select signaling pathways depend upon and localize to primary cilia, and second, inherited ciliary defects underlie pediatric syndromes that are now recognized as ciliopathies. These findings revealed the importance of primary cilia but also highlighted critical gaps in our understanding. Key questions include: how are cilia assembled, maintained, and disassembled; how do cilia promote signaling; how is cilium disassembly linked to the cell cycle; how are ciliary functions regulated in a cell type- and tissue-specific manner; and how do ciliary defects contribute to disease? At present, many ciliary regulators have yet to be identified or characterized in detail, and thus the answers to these questions remain elusive. The long-term goal of my research program is to understand the molecular mechanisms that underlie mammalian primary cilium function. We further aim to elucidate how ciliary functions are connected to other cellular processes and structures, including the cell cycle that governs cilium formation and the centrosome on which the cilium is built. Lastly, we seek to identify how ciliary defects contribute to disease, with the hope that such information can improve understanding, diagnosis, and treatment of associated pathologies. To achieve these goals, we combine our unique expertise in genome-wide functional screening with innovative biochemical and microscopy methods. Recently, we have conducted genome-wide CRISPR-based loss-of-function and gain-of-function screens to systematically identify positive and negative regulators of ciliary signaling, respectively. We further demonstrated the value of these approaches by establishing screen hits as new ciliary regulators, including Rab34 as a mediator of ciliogenesis, Ppp2r3c as a centriolar phosphatase subunit, and Sarm1 as part of a novel pathway for cilia disassembly. Additionally, these studies have provided new insights into the roles of ciliary and centriolar defects in disease. We now propose to build on these efforts through: 1) functional analysis of a newly identified pathway that mediates cell cycle-regulated cilia disassembly, 2) mechanistic studies on novel regulators of cilia biogenesis, including a transmembrane microprotein we find to be essential for ciliogenesis, 3) development of microscopy-based screening tools for high-throughput identification of ciliary regulators, and 4) application of these tools across diverse cell types and ciliary processes for systematic molecular analysis of primary cilium function. 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 disease. Lastly, the functional genomic tools developed here are anticipated to have broad utility to the cell biology community.