Microtubule-based machines are responsible for the determination of cell shape, the intracellular transport of organelles, the separation of chromosomes during cell division, and the beating of cilia and flagella. Defects in the function or regulation of the motor proteins that drive the microtubule-based machines result in changes in cell shape, misplacement of organelles, infertility, respiratory disease, and developmental detects. The long term goal of the proposed research is to understand the molecular mechanisms that regulate the assembly, targeting, and activity of the dynein family of motor proteins. We have identified several new genes that are involved in the assembly and regulation of the dynein motors in Chlamydomonas. We will further characterize these genes and gene products to identify the components that interact to position a dynein motor complex correctly inside the cell and regulate its activity. The proposed experiments will capitalize on the highly ordered structural organization of the flagellar axoneme and the ease of genetic analysis in Chlamydomonas to ask how a single cell controls the assembly and activity of its multiple flagellar dyneins.
Our specific aims are: (1) To identify novel genes that encode components involved in the targeting and regulation of the I1 inner arm dynein. Genetic strategies will be used to isolate mutations in a dynein intermediate chain implicated in the regulation of the I1 dynein and to identify novel components in regulatory pathway. (2) To determine the subcellular distribution and function of the dynein regulatory components. Biochemical methods and immunolocalization procedures will be used to analyze the physical relationship between the novel regulatory polypeptides and the associated dynein motors. (3) To identify the polypeptides associated with an unusual cytoplasmic dynein (cDhc1 b) required for retrograde intraflagellar transport (IFT) and flagellar assembly. Biochemical and molecular strategies will be used to identify subunits of the cDhc1b complex. Mutations that alter the distribution and activity of cDhc1b motor will also be studied to identify interacting polypeptides. The results will provide basic information about the organization of dynein motors and associated regulatory components in the flagellar axoneme and new insights into the fundamental mechanisms that target the dynein family of motors to specific subcellular locations and regulate their activity. Given the growing awareness of the critical role played by motor proteins in a wide range of human diseases, these studies will also have important implications for both diagnostic and therapeutic strategies in the treatment of human health.
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