Integrating functional proteomics, genome engineering, and live-cell microscopy to the study of ciliopathies - Project Summary
The goal of this project is to determine how ciliopathy-related mutations contribute to ciliary
pathophysiology. Ciliopathies are disorders rooted in ciliary dysfunction and exhibit overlapping clinical
features, including developmental delay, intellectual disability, polydactyly, retinal dystrophy, and progressive
involvement of the kidney and liver. While individually rare, ciliopathies combined affect 1/500 individuals and
each of the ~30 distinct ciliopathies is caused by dysfunction of a specific protein network related to the cilium,
although the precise cellular mechanisms remain elusive. The primary cilium is an antenna-like projection
found on nearly every cell; it extends from the cell body, where it receives and interprets signals, thus allowing
cells to respond to their environment. Cilia are partitioned from the cellular cytoplasm by the transition zone
that regulates protein trafficking. A dedicated active transport system, intraflagellar transport, moves proteins
bound for the cilium across this barrier and works in conjunction with multiple methods for protein retention and
selective egress. The proteins involved in this selective protein transport are implicated in a range of
ciliopathies, indicating that aberrant ciliary protein content likely contributes to the etiology of these disorders.
I will use my novel human cilia isolation protocol and state-of-the-art mass spectrometry approach to
assess global ciliary protein composition, defining differences between controls and cells harboring ciliopathy-
associated hypomorphic mutations. This work will provide a comprehensive, unbiased catalog of mislocalized
proteins in Joubert (K99) and Bardet-Biedl (R00) syndromes, thus providing a rich resource for future work to
dissect the protein networks involved in ciliopathies. In a complementary approach, I will determine how
ciliopathy-associated mutations affect the dynamics of protein trafficking by endogenously tagging key
ciliopathy proteins and following their movement using live-cell microscopy. This work will answer critical
questions about the impact of ciliopathy-associated mutations on entry into, retention within, and exit from cilia.
This application proposes innovative techniques that are easily extendable to other proteins/ciliopathies, and
importantly, investigates protein content and dynamics in the human disease context rather than with null
mutations in animal models. Together, this work will shed light on the etiology of ciliopathies and catalyze the
development of future therapies.
