Tetrahymena thermophila contains a conventional γ-tubulin that is differentially required for the maintenance of different microtubule-organizing centers

Abstract

The gene (GTU1) encoding Tetrahymena thermophila γ-tubulin was cloned and analyzed. GTU1 is a single-copy, essential gene encoding a conventional γ-tubulin. HA-tagged GTUlp localizes to four microtubule-organizing centers (MTOCs) in vegetative cells: basal bodies (BBs), macronuclear envelopes, micronuclear envelopes, and contractile vacuole pores. γ-Tubulin function was studied by placing the GTU1 gene under control of an inducible-repressible promoter. Overexpression of GTU1 had no detectable effect on cell growth or morphology. Depletion of γ-tubulin resulted in marked changes in cell morphology and in MT bundling. MTOCs showed different sensitivities to γ-tubulin depletion, with BBs being the most sensitive. γ-Tubulin was required not only for the formation of new BBs but also for maintenance of mature BBs. BBs disappeared in stages, first losing γ-tubulin and then centrin and glutamylated tubulin. When GTU1 expression was reinduced in depleted cells, BBs reformed rapidly, and the normal, highly organized structure of the Tetrahymena cell cortex was reestablished, indicating that the precise patterning of the cortex can be formed de novo.