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.
The primary cilium is an antenna-like cellular protrusion that allows cells to respond to external signals that instruct cell growth, differentiation, and proliferation. Aberrant ciliary signaling causes pediatric syndromes and can promote cancer development, while imbalances in cilium assembly versus disassembly are commonly observed in tumors and may contribute to uncontrolled cell growth. This proposal aims to improve our understanding of the roles of primary cilia in health and disease by elucidating the molecular pathways that underlie cilium formation, function, and disassembly.
