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.
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.
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