This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ciliate Tetrahymena is covered with more than 1000 basal bodies. Each basal body is composed of 9 triplet microtubules in a circular array, very similar in its organization to the centrioles in centrosomes. Also similar to centrioles, basal bodies are assembled from a specific site at the cellular end of an existing basal body. We have initiated an analysis of Tetrahymena basal body duplication as a model for centriole duplication. As part of this project, we have started electron tomographic analysis of basal bodies and their assembly intermediates in Tetrahymena. We have prepared Tetrahymena cells by chemical fixation and by high-pressure freezing and freeze-substitution. The basal bodies have excellent morphology with both these fixations, though some of the frozen cells show ice damage. A few preliminary tomograms of basal bodies in mutant and wild-type cells have been constructed to compare the two sample types. We have also done tomograms of basal bodies in pellicles. Pellicles are cell ghosts resulting from mechanical disruption of the cells and extraction of the cytoplasm. It has been previously reported that basal bodies remain intact in pellicles, and we have repeated that result. We are comparing the tomograms of basal bodies in intact cells and in pellicles, seeking any structural differences. We are using basal bodies in pellicles for localization and biochemical studies and will need to correlate those results with our structural studies of fixed cells. Our future plans for tomography of basal bodies in Tetrahymena include the description of basal body duplication in wild-type and in mutant cells. This analysis will help identify which assembly processes or intermediates are missing in the mutants. Furthermore, we will complete our tomographic examination of the basal bodies in pellicles. If we can assemble basal bodies or their intermediates in vitro, those structures will be examined by tomography to compare them with their in vivo counterparts.
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