Cilium-associated structures in rod cells

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The goal of this Supplement request is to acquire a Typhoon 5 to enable the experiments planned for project R01-EY026545, Cilium-Associated Structures in Photoreceptors, as well as those for R01-EY031949 and other NEI-funded projects at Baylor College of Medicine. An instrument of this kind is essential for rigorous quantification of proteins, nucleic acids, lipids and other small molecules separated by gel electrophoresis or thin-layer chromatography. It will eliminate the need for outmoded x-ray film-based detection and allow routine rapid quantification of immunoblots, a workhorse technique for our NEI projects. A condensed summary of the parent grant follows. The goal of this project is to develop a thorough understanding of the structural and molecular basis of p function of the rod sensory cilium, and to understand the molecular mechanisms of rod cell death in ciliopathies. There are three Specific Aims: 1. Use cryo-electron tomography (cryo-ET) and recent developments in sub-tomogram averaging to determine the three-dimensional structure to nanometer resolution of repeating structures of the rod cell connecting cilium and basal body, including microtubule doublets and triplets, microtubule inner proteins, “Y-shaped links”, transition fibers and appendages. Our goal is to apply recent developments in hardware and software to rod cells in both wild type retinas and in animal models of retinal degeneration. 2. Use superresolution fluorescence to test hypotheses about trafficking of specific proteins and about the roles of IFT (intraflagellar transport) particles and the BBSome (a coat-forming protein complex implicated in the blinding ciliopathy, Bardet-Biedl syndrome) in ciliary trafficking in rods. Two- color superresolution fluorescence and quantitative interaction analysis will be used to assess putative interactions between IFT proteins or BBS proteins and outer segment membrane proteins, as well as well as proteins normally excluded from the outer segment which mis-accumulate there in BBS-deficient mice. These experiments will test the hypothesis that specific membrane proteins are actively trafficked through the connecting cilium membrane through their association with IFT particles, whereas others are transported via alternative routes and excluded proteins are actively removed by the BBSome. 3. Use mouse models to test the hypotheses that CEP290 is a major component of the “Y-shaped links” extending from the ciliary axoneme to the membrane, using superresolution fluorescence, conventional TEM, and cryo-electron tomography with timed gene disruption or gene restoration at different developmental stages to distinguish initiating as opposed to secondary events in the development of the pathophysiology of ciliopathies associated with this protein