Date of Award
Doctor of Philosophy (PhD)
Tin Tin Su
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.
Ross, Ian, "ε-tubulin and δ-tubulin in Tetrahymena thermophila Are Essential Components of Basal Bodies" (2012). Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations. 55.