We study the organization and function of microtubules with genetic, biochemical, cytological, and molecular techniques. In a Chlamydomonas cell there are five kinds of microtubules that play roles in flagellar motility, cellular asymmetry, cytokinesis, and chromosome segregation. We are interested in how basal bodies or centrioles interact with other components of the centrosome in the assembly and function of these microtubules. Three classes of mutations will be analyzed in this work. We are interested in using mutations to define components of the centrosome and its role in the cell cycle. We have identified mutations at two loci, FLA10 and APM1, that are an excellent entry point into this question. Specific alleles at these loci interact to produce a cold-sensitive synthetic lethal phenotype; these mutations block the ability of cells to complete the cell cycle. Intriguingly, the lethality of these double mutant strains is rescued by the absence of basal bodies. One hypothesis suggest that these genes identify components of the basal body and the surrounding pericentriolar material, which are the two components of the centrosome. We will use molecular techniques to isolate and characterize these genes as well as genetic studies to identify other genes that interact with these loci. Although we have shown using the bald2 mutation in Chlamydomonas that basal bodies (and centrioles) are largely nonessential for proper chromosome segregations and progression through the cell division cycle, they appear to be required for the fidelity of cytokinesis, flagellar assembly, and cellular asymmetry. It is likely that these processes are affected because of the aberrant assembly of the rootlet microtubules, which are a specialized class of microtubules. We have identified several new mutations, which have assembled basal bodies when assayed by immunofluorescence, have altered rootlet microtubules. These cells have defects in cytokinesis, flagellar assembly, and cellular asymmetry. These mutations will be studied by structural, molecular, and genetic techniques. We are also interested in using mutations to define the mechanisms by which the identity of basal bodies is established, which may play a role in generating asymmetry in the cell. Screens for this class of mutants are underway.
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