Date of Award

Spring 1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Mark Winey

Second Advisor

Tin Tin Su

Third Advisor

Andy Hoenger

Fourth Advisor

Jennifer DeLuca

Abstract

Basal bodies and centrioles are conserved microtubule-based organelles whose improper assembly leads to a number of diseases, including ciliopathies, such as polycystic kidney disease and Bardet-Biedl syndrome, and cancer. Tubulin family members are conserved components of these structures that are integral to their proper formation and function. The nine-fold triplet microtubule organization of basal bodies is a widely conserved structural feature. Two proteins that have been implicated in the proper assembly and maintenance of this structure are ε- and δ-tubulin. I sought to ask what the functions of these two proteins are in the assembly and maintenance of the core microtubule triplets of the basal body in Tetrahymena thermophila.

I have identified the ε-tubulin and δ-tubulin genes genes in Tetrahymena. I have localized ε-tubulin through immunofluorescence to basal bodies. Immuno-electron microscopy has shown that ε-tubulin localizes primarily to the core microtubule scaffold. Complete genomic knockouts of ε-tubulin and δ-tubulin revealed that each gene is essential for viability. ε-tubulin is required for the assembly and maintenance of the triplet microtubule blades of basal bodies. I have conducted site-directed mutagenesis of the ε-tubulin gene and shown that residues within the nucleotide-binding domain, longitudinal interacting domains, and C-terminal tail are required for proper function. A single amino acid change of Thr150, a conserved residue in the nucleotide-binding domain, to Val is a conditional mutation that results in defects in the spatial and temporal assembly of basal bodies as well as their stability. I have genetically separated functions for the domains of ε-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.

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