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 newgenes involved in the assembly and regulation of dynein motors in Chlamydomonas. We will continue tocapitalize on the highly ordered structural organization of the flagellar axoneme and the ease of geneticanalysis in Chlamydomonas to further characterize these genes and gene products and identify interactingcomponents that regulate dynein activity.
Our specific aims are: (1) To identify the location and function ofeach 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 todetermine the position of each subunit within the axoneme. Biochemical procedures and in vitro slidingassays will also be used to assess the impact of mutations in IC138 phosphorylation sites. Flagellarwaveforms of mutant cells will be analyzed by high speed digital imaging. (2) To identify and characterizeinteractions between components of a dynein regulatory complex (DRC). We have demonstrated that theDRC is an integral part of the nexin link, and we have characterized three DRC subunits as highlyconserved, coiled coil proteins required for assembly of the DRC and associated DHCs. Epitope-taggedconstructs 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 characterizecomponents that interact with a cytoplasmic dynein required for retrograde intraflagellar transport (IFT). Wehave identified a novel DIG associated with the retrograde motor. RNA interference and GFP taggedsubunits will be used to characterize its role in intraflagellar transport. We have also identified the FLA4gene product as a conserved TPR repeat protein implicated in human disease and nervous systemdevelopment. Specific antibody probes and epitope tagged constructs will be used to analyze its subcellulardistribution and determine its role in both IFT and flagellar assembly. The studies will provide basicinformation about the organization of dyneins and associated regulatory components in the axoneme andnew 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 oforganelles, chromosome separation during mitosis, and beating of cilia and flagella. Defects in the functionof the motor proteins that drive these machines result in defects in nervous system development andfunction, 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 willhave 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 #
4R37GM055667-14
Application #
7789203
Study Section
Special Emphasis Panel (NSS)
Program Officer
Gindhart, Joseph G
Project Start
1997-04-01
Project End
2015-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
14
Fiscal Year
2010
Total Cost
$458,096
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Chien, Alexander; Shih, Sheng Min; Bower, Raqual et al. (2017) Dynamics of the IFT machinery at the ciliary tip. Elife 6:
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

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