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 e-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.
Original language | English (US) |
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Pages (from-to) | 3441-3451 |
Number of pages | 11 |
Journal | Journal of Cell Science |
Volume | 126 |
Issue number | 15 |
DOIs | |
State | Published - Sep 9 2013 |
Externally published | Yes |
Keywords
- Basal body
- Epsilon tubulin
- Microtubule organizing center
- Tetrahymena thermophila
ASJC Scopus subject areas
- Cell Biology