Primary cilia organize signaling pathways such as vision, olfaction and the Hedgehog developmental pathway inside a specialized environment. The soluble contents and the membrane of the cilium are separated from the rest of the cell by diffusion barriers, thus endowing cilia with a unique physico-chemical environment. Depending on the status of Hedgehog signaling, specific signaling intermediates move into or out of cilia and it is predicted that the ciliary composition is modified by Hedgehog signaling. However, it has not been possible to assess the extent of the changes in the ciliary proteome upon Hedgehog pathway activation because cilia cannot be readily isolated from mammalian cells. Dysfunction of primary cilia is the underlying cause of a collection of hereditary disorders named ciliopathies characterized by obesity, kidney cysts, skeletal malformations and sensory defects. Obtaining a catalogue of primary cilium proteins and comparing the ciliary proteome of ciliopathy to healthy cells would provide a rapid and effective way to characterize ciliopathies. Thus, proteomics of primary cilia is currently a major technical blockage in the discovery of novel ciliary mechanisms. Rather than isolating cilia, we sought to attach the small molecule biotin to all ciliary protein by targeting a biotinylation enzyme to the primary cilium. This approach named proximity labeling has shown great promise in obtaining the proteome of mitochondria. We present promising preliminary data showing the validity of this approach for ciliary proteomics and wish to apply it to compare the ciliary proteome between different signaling states or between ciliopathy mutants and healthy cells. The results obtained by comparative ciliary proteomics will provide unique insights into several important questions and generate testable molecular models. The proposal is intended to develop a promising technology and to serve as a stepping-stone towards a rapid and effective comparative ciliary proteomics platform.

Public Health Relevance

Anomalies of the primary cilium underlie the etiology of a class of disorders presenting with obesity, skeletal abnormalities, kidney cysts and retinal degeneration. The proposal aims to establish a novel method to systematically identify the alterations in the protein composition of cilia between healthy individuals and ciliopathy patients This innovative technological platform has the potential to reveal unsuspected pathomechanisms that may lead to novel therapeutic interventions.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
7R21HD087126-03
Application #
9590675
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Toyama, Reiko
Project Start
2015-12-01
Project End
2018-11-30
Budget Start
2017-09-01
Budget End
2018-11-30
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94118
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Ye, Fan; Nager, Andrew R; Nachury, Maxence V (2018) BBSome trains remove activated GPCRs from cilia by enabling passage through the transition zone. J Cell Biol 217:1847-1868
Steinman, Jonathan B; Santarossa, Cristina C; Miller, Rand M et al. (2017) Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action. Elife 6:
Lee, Tina H; Nachury, Maxence V (2017) Membrane traffic control by cytoskeletal and molecular machines. Mol Biol Cell 28:697-698