γ-Tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation

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Abstract

γ-TUBULIN is a newly identified member of the tubulin family whose sequence is highly conserved from yeast to man. This minor microtubule protein is localized to the microtuble organizing centres1–3 and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans4,5. Here we investigate the in vivo function of γ-tubulin in mammalian cells using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of γ-tubulin. After microinjection into cultured mammalian cells, immunofluorescence localization revealed that this antibody binds to native centrosomes at all phases of the cell cycle. In the presence of the γ-tubulin antibody, microtubules fail to regrow into cytoplasmic arrays after depolymerization induced by nocodazole or cold. Furthermore, cells injected immediately before or during mitosis fail to assemble a functional spindle. Thus in vivo γ-tubulin is required for microtubule nucleation throughout the mammalian cell cycle.

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