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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM055667-16
Application #
8287052
Study Section
Special Emphasis Panel (NSS)
Program Officer
Gindhart, Joseph G
Project Start
1997-04-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
16
Fiscal Year
2012
Total Cost
$433,858
Indirect Cost
$146,535
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Alford, Lea M; Stoddard, Daniel; Li, Jennifer H et al. (2016) The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme. Cytoskeleton (Hoboken) 73:331-40
Reck, Jaimee; Schauer, Alexandria M; VanderWaal Mills, Kristyn et al. (2016) The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas. Mol Biol Cell 27:2404-22
Austin-Tse, Christina; Halbritter, Jan; Zariwala, Maimoona A et al. (2013) Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia. Am J Hum Genet 93:672-86
Wren, Kathryne N; Craft, Julie M; Tritschler, Douglas et al. (2013) A differential cargo-loading model of ciliary length regulation by IFT. Curr Biol 23:2463-71
Bower, Raqual; Tritschler, Douglas; Vanderwaal, Kristyn et al. (2013) The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes. Mol Biol Cell 24:1134-52
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
O'Toole, Eileen T; Giddings Jr, Thomas H; Porter, Mary E et al. (2012) Computer-assisted image analysis of human cilia and Chlamydomonas flagella reveals both similarities and differences in axoneme structure. Cytoskeleton (Hoboken) 69:577-90
Heuser, Thomas; Barber, Cynthia F; Lin, Jianfeng et al. (2012) Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein. Proc Natl Acad Sci U S A 109:E2067-76
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
VanderWaal, Kristyn E; Yamamoto, Ryosuke; Wakabayashi, Ken-ichi et al. (2011) bop5 Mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric waveforms. Mol Biol Cell 22:2862-74

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