Molecular and Genetic Analysis of Flagellar Dyneins

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DESCRIPTION (provided by applicant): The motility of eukaryotic cilia and flagella is known to depend on dynein ATPase motors that move along doublet microtubules. Flagellar dyneins must be regulated to coordinate their activity during normal bend formation and propagation, and additionally to modulate beat frequency and waveform in response to changing physiological requirements. Two genes essential for outer row dynein assembly, ODA7 and ODA8, have recently been identified as homologs of LC1, which binds to the HC gamma dynein catalytic domain and may act as a regulatory light chain. Hypothesized functions of Oda7p and Oda8p as HC alpha and HC beta regulatory light chains will be tested. Scaffold proteins have been hypothesized to function as links between multiple dynein isoforms along each doublet microtubule. Two candidate scaffold protein genes (PF13 and ODA16) have been cloned based on insertional mutations that disrupt dynein assembly and function. Experiments are proposed to define the role of each gene product in docking dyneins to regulatory proteins on the doublet microtubule surface. Kinesins represent another family of microtubule associated motor proteins found in flagella, but their role in flagellar motility is not known. Global regulation of flagellar motility involves signals transmitted by radial spokes between doublet-associated dyneins and the central pair microtubule-associated complex. The role of two central pair projections in this process will be studied using a mutation that disrupts one projection (cpcl) and RNAi-mediated knockdown of a kinesin found in another projection (Klp1). Work on central pair kinesins will be extended to include all kinesins identified as central pair proteins, and knockdown strains will be used to screen for null mutations in central pair kinesin genes.