Ciliopathies are genetic deficiencies in proteins important for structure and function of primary (non-motile) cilia. They give rise to a range of defects, depending on the alleles involved, including kidney cysts, obesity, male infertility, polydactyly, and mental retardation. The light-sensing outer segments of retinal rod and cone photoreceptors are specialized primary cilia, and one of the most frequent symptoms of ciliopathies is degeneration of the retina, leading to blindness. This project aims to determine the structural basis of ciliopathies by determining with unprecedented detail and accuracy the three-dimensional architecture of the structures at the base of the outer segments most directly affected by ciliopathies. These include the connecting cilium or transition zone, the basal body complex, and associated filaments, fibers, and vesicles. The structures will be determined in cells from normal animals and animal models of retinal ciliopathies. The structures of large macromolecular complexes essential for cilium formation and/or function, formed by BBS proteins (named for the involvement of the proteins that form it in a type of ciliopathy known as Bardet-Biedl Syndrome, BBS), will also be determined for normal and diseased retinas. The major techniques used will be cryo-electron microscopy and super-resolution fluorescence microscopy, including single particle analysis and the emerging technique of cryo-electron tomography, along with computational averaging of many instances of the same sub-cellular objects to improve signal-to-noise ratios and extend resolution. These approaches will be supplemented with conventional fluorescence and transmission electron microscopy imaging, and scanning electron microscopy. Application of these approaches to mouse models of Bardet- Biedl syndrome will be used to test hypotheses about the roles of BBS proteins in formation and maintenance of ciliary structure, and about the pathophysiological mechanisms leading to cell death resulting from genetic deficiencies in BBS proteins.
The specific aims are: 1. Determine the structure of repeating features of rod cilia in unprecedented detail by cryo-electron tomography and sub-tomogram averaging, correlated with conventional transmission electron microscopy and super-resolution fluorescence microscopy. Accomplishing this aim will enable us and others to test hypotheses about the roles of specific proteins and genes in establishing and maintaining these structures. 2. Determine the structure of the Bardet-Biedl protein coat-forming complex, or BBSome to high resolution using recent advances in cryo-electron microscopy and single particle analysis. We will test the hypothesis that subunits of the BBSome assume structures and protein-protein interactions similar to those in other coat-protein complexes. 3. Use knock-out mouse models to test the hypothesis that defects in the BBSome lead to structural defects in cilium-associated features of rods as a secondary result of loss of BBSome functions deficiencies related to membrane protein sorting and/or trafficking.
This proposal is focused on understanding the structural link between genetic deficiencies and inherited disease. Doing so will help us understand the mechanisms of retinal neurodegeneration and other cilium-associated pathologies and could lead to new therapeutic approaches.
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