title: ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies.
abstract: Basal bodies and centrioles are conserved microtubule-based organelles the improper assembly of which leads to a number of diseases, including ciliopathies and cancer. Tubulin family members are conserved components of these structures that are integral to their proper formation and function. We have identified the ε-tubulin gene in Tetrahymena thermophila and detected the protein, through fluorescence of a tagged allele, to basal bodies. Immunoelectron microscopy has shown that ε-tubulin localizes primarily to the core microtubule scaffold. A complete genomic knockout of ε-tubulin has revealed that it is an essential gene required for the assembly and maintenance of the triplet microtubule blades of basal bodies. We 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. We 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.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Colorado-Boulder, Boulder, CO 80309, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Ross
authors.forename: Christina
authors.initials: C
authors.lastname: Clarissa
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Giddings
authors.forename: Mark
authors.initials: M
authors.lastname: Winey
mesh_terms: D064652:Basal Bodies / Q000378:metabolism / Q000502:physiology*; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002502:Centrioles / Q000235:genetics / Q000378:metabolism; D016770:Ciliophora Infections / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D016808:Tetrahymena thermophila / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D014404:Tubulin
keywords: Basal body; Epsilon tubulin; Microtubule organizing center; Tetrahymena thermophila