The long-term goal of the proposed research is to understand the mechanisms that regulate the assembly, targeting, and activity of the dynein family of motors in cilia and flagella. We have identified several new genes involved in the assembly and regulation of dynein motors in Chlamydomonas. We will continue to capitalize on the highly ordered structural organization of the flagellar axoneme and the ease of genetic analysis in Chlamydomonas to further characterize these genes and gene products and identify interacting components that regulate dynein activity.
Our specific aims are: (1) To identify the location and function of each subunit within the II dynein complex. We have characterized an IC/LC sub-complex that is required for 1 activity and regulation, but not for II assembly. High resolution electron microscopy will be used to determine the position of each subunit within the axoneme. Biochemical procedures and in vitro sliding assays will also be used to assess the impact of mutations in IC138 phosphorylation sites. Flagellar waveforms of mutant cells will be analyzed by high speed digital imaging. (2) To identify and characterize interactions between components of a dynein regulatory complex (DRC). We have demonstrated that the DRC is an integral part of the nexin link, and we have characterized three DRC subunits as highly conserved, coiled coil proteins required for assembly of the DRC and associated DHCs. Epitope-tagged constructs and specific antibody probes will be used to further define the biochemical properties of the DRC, and co-immunoprecipitation and comparative proteomics will be used to identify other DRC components. The role of the DRC in the regulation of microtubule sliding will also be assessed. (3) To characterize components that interact with a cytoplasmic dynein required for retrograde intraflagellar transport (IFT). We have identified a novel DIG associated with the retrograde motor. RNA interference and GFP tagged subunits will be used to characterize its role in intraflagellar transport. We have also identified the FLA4 gene product as a conserved TPR repeat protein implicated in human disease and nervous system development. Specific antibody probes and epitope tagged constructs will be used to analyze its subcellular distribution and determine its role in both IFT and flagellar assembly. The studies will provide basic information about the organization of dyneins and associated regulatory components in the axoneme and new insights into the mechanisms that target the dyneins to specific locations and regulate their activities.
Microtubule-based machines are responsible for the determination of cell shape, intracellular transport of organelles, chromosome separation during mitosis, and beating of cilia and flagella. Defects in the function of the motor proteins that drive these machines result in defects in nervous system development and function, infertility, chronic respiratory disease, polycystic kidney disease, and other developmental defects. Given the critical roles played by cilia and flagella in a wide range of ciliopathies, the proposed studies will have important implications for diagnostic and therapeutic strategies in the treatment of human disease.
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|Wirschell, Maureen; Olbrich, Heike; Werner, Claudius et al. (2013) The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans. Nat Genet 45:262-8|
|Lin, Jianfeng; Tritschler, Douglas; Song, Kangkang et al. (2011) Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella. J Biol Chem 286:29175-91|
|Nicastro, Daniela; Fu, Xiaofeng; Heuser, Thomas et al. (2011) Cryo-electron tomography reveals conserved features of doublet microtubules in flagella. Proc Natl Acad Sci U S A 108:E845-53|
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|Toba, Shiori; Fox, Laura A; Sakakibara, Hitoshi et al. (2011) Distinct roles of 1alpha and 1beta heavy chains of the inner arm dynein I1 of Chlamydomonas flagella. Mol Biol Cell 22:342-53|
|Dentler, William; Vanderwaal, Kristyn; Porter, Mary E (2009) Recording and analyzing IFT in Chlamydomonas flagella. Methods Cell Biol 93:145-55|
|Heuser, Thomas; Raytchev, Milen; Krell, Jeremy et al. (2009) The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella. J Cell Biol 187:921-33|
|Bower, Raqual; VanderWaal, Kristyn; O'Toole, Eileen et al. (2009) IC138 defines a subdomain at the base of the I1 dynein that regulates microtubule sliding and flagellar motility. Mol Biol Cell 20:3055-63|
|Ikeda, Kazuho; Yamamoto, Ryosuke; Wirschell, Maureen et al. (2009) A novel ankyrin-repeat protein interacts with the regulatory proteins of inner arm dynein f (I1) of Chlamydomonas reinhardtii. Cell Motil Cytoskeleton 66:448-56|
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