# Mechanisms of Primary Cilium Assembly and Disassembly

> **NIH NIH R35** · YALE UNIVERSITY · 2022 · $418,750

## Abstract

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 cil...

## Key facts

- **NIH application ID:** 10430232
- **Project number:** 5R35GM137956-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** David King Breslow
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $418,750
- **Award type:** 5
- **Project period:** 2020-09-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10430232

## Citation

> US National Institutes of Health, RePORTER application 10430232, Mechanisms of Primary Cilium Assembly and Disassembly (5R35GM137956-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10430232. Licensed CC0.

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