epsilon-tubulin and delta-tubulin in Tetrahymena thermophila are essential components of basal bodies

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 epsilon- and delta-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 epsilon-tubulin and delta-tubulin genes in Tetrahymena. I have localized epsilon-tubulin through immunofluorescence to basal bodies. Immuno-electron microscopy has shown that epsilon-tubulin localizes primarily to the core microtubule scaffold. Complete genomic knockouts of epsilon-tubulin and delta-tubulin revealed that each gene is essential for viability. epsilon-tubulin is required for the assembly and maintenance of the triplet microtubule blades of basal bodies. I have conducted site-directed mutagenesis of the epsilon-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 epsilon-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.