Proteomics of Primary Cilia through Proximity Labeling

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 DESCRIPTION (provided by applicant): 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.