Dynein regulation and flagellar asymmetry in Chlamydomonas reinhardtii

The eukaryotic flagellum is a complex organelle that aligns and regulates dynein molecules so that they will work as a unit to generate motion. The dynein molecules in the flagellum must be regulated in asymmetric fashions, both along the length and around the circumference of the flagella in order to generate a waveform. In Chlamydomonas reinhardtii, asymmetries between the two flagella are likely to be used in the coordinated turning during phototactic behavior. This thesis attempts to identify and characterize the regulation of dynein activity, especially in relation to flagellar asymmetries. I hypothesized that at least one of the Chlamydomonas dynein complexes would be regulated in an asymmetric fashion between the two flagella during phototaxis. To test this hypothesis, I characterized mutant strains that were unable to perform phototaxis with the goal of identifying axonemal components involved in dynein regulation. A large collection of phototactic mutant strains were screened for additional phenotypes that would be expected of strains with dynein regulation defects. Several of these strains were selected for further analyses.; Three strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Fractionated dynein from the mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138 kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mial-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138 kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which assemble only one of the two flagella, also contained relatively high levels of the altered phosphorylation forms of the 138 kD intermediate chain. These data suggest that the f dynein complex is required for phototactic behavior and that the complex may be phosphoregulated asymmetrically between the two flagella.