The goal of the research proposed here is to exploit the unique advantages of the ciliated alga Chlamydomonas to functionally characterize the ciliary protein CEP290/nephrocystin-6 (hereon referred to as CEP290). Mutations in CEP290 cause cilia-related human diseases, including Leber congenital amaurosis, Meckel syndrome, Bardet-Biedl syndrome (BBS), Joubert syndome, and Senior-Loken syndrome. Although disease manifestation varies in these disorders, all of these disorders present with characteristics that have been linked to defects in cilia: cystic kidney disease or nephronophthisis, retinal degeneration and blindness, polydactyly, defects in the central nervous system, liver fibrosis, and in the case of BBS,obesity. Nephronophthisis (NPHP), which results from mutations in any of nine distinct genes (nephrocystins, NPHP1-9), is the most common genetic cause of renal failure in children and young adults. It is currently unknown how defects in CEP290 affect cilia function and lead to cilia-related diseases. Evidence in multiple organisms, including Chlamydomonas, suggests that CEP290 localizes to the transition zone of cilia and flagella-a structurally complex region about which very little is known. These studies will test the hypothesis that CEP290 localizes to the transition zone where it regulates the movement of proteins into the flagella. This will be tested by biochemical, genetic, and cell biological analysis of ciliary phenotypes present in a Chlamydomonas insertional mutant harboring a large deletion of the CEP290 gene. The precise localization of CEP290 will be determined using biochemical, genetic, and morphological approaches. The protein composition of CEP290 mutant flagella will be compared to that of wild type using 2D gel electrophoresis and mass spectrometry. The outstanding biochemistry that Chlamydomonas offers will be utilized to purify complexes containing CEP290, and proteins co-purifying with CEP290 will be identified by mass spectrometry. Although nephrocystin proteins display common localization patterns and defects in the nephrocystin genes all result in NPHP, it is unknown if the nephrocystins interact with each other or regulate each other's localization. This limitation will be addressed using another Chlamydomonas insertional mutant that is null for nephrocystin-4. This research has great potential to add insight into the basic function of CEP290, determine how CEP290 defects affect cilia structure and/or function, reveal novel functions for the transition zone, and provide new information on the basic biology of cilia, and is therefore of utmost interest and relevance to public health.
| Craige, Branch; Tsao, Che-Chia; Diener, Dennis R et al. (2010) CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J Cell Biol 190:927-40 |
