The long-term goals are to understand the assembly and function of cilia and flagella. Most of the research is being carded out using Chlamydomonas, because its flagella are readily studied by a combination of genetic, molecular genetic, biochemical, and cell biological approaches. Additional studies are being carded out in the mouse. Biochemical studies will test the hypothesis that the recently identified ODA5 protein is part of a previously unknown complex that is necessary for assembly of the outer dynein arm onto the Chlamydomonas flagellar axoneme. If so, cDNAs encoding the other components of the complex will be isolated and sequenced, and mutants with defects in these genes will be identified and characterized. Intraflagellar transport (IFT), which moves particles from the cell body to the tip of the flagellum and then back to the cell body, is important for the assembly of all cilia and flagella. Chlamydomonas cytoplasmic dynein lb, the retrograde IFT motor, will be analyzed to determine its subunit composition, and cDNAs encoding newly identified subunits will be cloned. Pulse-label studies will determine if any subunits are phosphorylated, which would suggest a role in the control of dynein lb. lmmunofluorescence microscopy of mutants expressing modified forms of dynein lb's light intermediate chain, D lbLIC, will be carded out to elucidate the roles of D lbLIC' s P-loop and phosphorylation sites. Chlamydomonas insertional mutants with reduced levels of dynein lb will be characterized to identify the defective genes, which may encode previously unidentified subunits of dynein lb. Mutants with defects in the IFFparticle proteins IFF20, IFF46, and IFF57 will be studied to understand the functions of these proteins. IFF particles from the mouse testis will be isolated and characterized to determine their composition, structure, and cargo. The epithelial cells lining the follicles of the thyroid gland have well-developed primary cilia, but the function of these cilia is unknown. Thyroids of Tg737 xk mice, which are defective in IFT, will be examined by scanning electron microscopy to determine if they fail to assemble normal primary cilia, and sera of mutant mice will be assayed to determine if thyroid hormone levels are abnormal. If so, the mutant thyroids will be studied by histology and electron microscopy to understand how the lack of primary cilia affects thyroid function. The studies promise to increase our understanding of a wide range of human diseases, including primary ciliary dyskinesia, in which the dynein arms are frequently missing, male infertility, and thyroid disorders.
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