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Awais Zia, Jacob Nellissery, Rivka Rachel, Anand Swaroop; CEP290 Localizes to Photoreceptor Connecting Cilium and Interacts with Centrosomal-cilia Proteins. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1807.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in CEP290 account for up to 25% of Leber congenital amaurosis (LCA). Cep290rd16 mice, which serve as a model for human LCA, harbor an in-frame deletion of Cep290 exons 35 - 39, leading to a truncated protein and resulting in rapid retinal degeneration. We hypothesized that CEP290 is part of a multi-protein complex required for structural and functional integrity of the primary cilium. The goal of this project is to localize CEP290 protein in photoreceptors and identify its potential interacting partners.
Antibodies were raised against two CEP290 peptides (N- and C-terminal) and tested by immunoblotting and immunohistochemistry in wild type, Cep290-knockout and Cep290rd16 mouse retina, and in IMCD3 cultured cells. CEP290 interacting proteins were identified by co-immunoprecipitation (co-IP) followed by mass spectrometry analysis.
Immunoblot analysis identified a 290 kDa CEP290 protein in wild-type mouse retina. In Cep290rd16 retina, a smaller band representing the truncated CEP290 protein of ~240 kDa was detected. In the wild type retina, the localization of CEP290 was primarily restricted to the connecting cilium of photoreceptors, distal to the basal body but not extending into the axoneme. Co-labeling of CEP290 with alpha or gamma tubulin as markers for cilium and basal body, respectively, further refined the CEP290 localization. Interestingly, CEP290 is restricted to basal body in IMCD3 cells. Mass spectrometry analysis of CEP290-immunoprecipitated proteins from retinal extracts has revealed several centrosomal and cilia proteins; these interactions are being validated by other methods.
Our studies strongly advocate a role of CEP290 in transport of proteins crucial in the biogenesis and/or maintenance of photoreceptor outer segments.
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