Flagellar Motility and Assembly

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Cilia and flagella are essentially identical cell organelles that have important roles in human health; as a result, defects in ciliary proteins cause human diseases, termed “ciliopathies.” The long-term goals of this research are to understand the structure, assembly, and function of these organelles. The studies will utilize Chlamydomonas and mice as model organisms, and will concentrate on processes and proteins that are highly conserved among ciliated organisms. A combination of genetic, biochemical, and cell biological approaches will be taken. Investigations will focus on three related areas of particular importance for understanding the basic biology of cilia and ciliopathies. First, experiments will determine the functions and specific locations within the cilium of uncharacterized ciliary proteins. The cilium contains over 650 proteins, of which fewer than half have been well characterized. The central pair of axonemal microtubules is apt to be particularly rich in uncharacterized proteins, so initial efforts will examine it. A second focus will be on the fundamental mechanism of intraflagellar transport (IFT), which is the movement of large, multi-subunit “trains” from the base of the cilium to the ciliary tip and then back to the cell body. These trains are made up of complexes including IFT-A and IFT-B, which carry cargos necessary for the assembly and maintenance of the cilium. Train formation in the cell body involves recruitment of IFT-A and IFT-B to the base of the cilium, loading of cargo onto the complexes, attachment of motors to the complexes, and injection of the completed train into the cilium. When this process is defective, ciliary assembly fails, but little is known about the individual steps in this process. Studies will use high-resolution structured-illumination microscopy and mutants in which train formation is arrested at various steps to determine the order of these steps and the roles of individual proteins in key parts of the process. Related studies will explore the specific function of IFT-A in ciliary assembly. In addition, single-particle cryo-electron microscopy will be carried out to determine the structure of IFT-A and IFT-B, which will be important for understanding how these complexes are arranged in the trains. A third focus will be on the transition zone, a specialized region between the basal body and the ciliary axoneme. The transition zone acts as a barrier that, in concert with IFT, is important for establishing and maintaining the protein content of cilia. However, the transition zone is still largely a “black box.” Mutants with defects in transition zone proteins will be investigated to learn more about the specific roles of these proteins in transition zone function and assembly, and to determine the composition of the highly conserved Y-links, which connect the transition zone microtubules to the overlying membrane and are critical to the transition zone's barrier function. The results will fill major gaps in our knowledge of cilia and flagella, and provide new insight into why defects in specific ciliary proteins cause human disease.