Flagellar proteins regulating motility, assembly and photobehavior in Chlamydomonas reinhardtii

University of Minnesota Ph.D. dissertation. July 2011. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor:Dr. Mary Porter. 1 computer file (PDF); vi, 195 pages, appendix A.Cilia and flagella are microtubule-based organelles that perform critical functions in human health and development. The I1 inner arm dynein and the IC138 subunit play a key role in the regulation of flagellar motility. To understand how the IC138 protein and its associated subunits modulate I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles in Chlamydomonas reinhardtii. We first characterize a mutation (bop5-2) that disrupts an IC138 protein sub-complex located at the base of the I1 inner arm dynein. We found the bop5-2 deletion also affects the Tubby-1 (TBY1) gene. To characterize TBY1's activity, tagged versions of TBY1 were transformed into bop5-2. TBY1 protein localizes to a unique ring shaped structure found between the two contractile vacuoles and within the nucleo-flagellar apparatus. The bop5-3, bop5-4 and bop5-5 strains, like other I1 mutants, swim forwards with reduced swimming velocities and display an impaired reversal response during photoshock. However, unlike mutants lacking the entire I1 complex, bop5 strains exhibit normal phototaxis. Analysis of the bop5-3 flagellar waveform reveals that loss of the IC138 sub-complex reduces shear amplitude, sliding velocities, and the speed of bend propagation. The results indicate that the IC138 sub-complex is necessary to generate an efficient waveform for optimal forwards and backwards motility, but it is not essential for phototaxis. Assembly and maintenance of eukaryotic cilia and flagella requires the conserved, bidirectional movement of protein complexes along the length of the axoneme known as intraflagellar transport (IFT). We characterize the function of various components of the IFT complex responsible for the retrograde transport of particles towards the cell body. We quantify the defects in retrograde IFT and flagella assembly observed in a series of mutants of the retrograde complex subunit LIC. We also analyze the expression and distribution of retrograde IFT components in a family of flagellar assembly mutants known as fla, and attempt to correlate these patterns with defects in IFT parameters and other behavioral phenotypes. We provide new evidence that defects in IFT motors can alter photoshock and phototaxis behaviors