Assembly , Targeting, and Regulation of Dynein Motors

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.