GAMMA-TUBULIN IS A CENTROSOMAL PROTEIN REQUIRED FOR CELL CYCLE-DEPENDENT MICROTUBULE NUCLEATION

Gamma-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 centres 1-3 and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans 4,5. Here we investigate the in vivo function of gamma-tubulin in mammalian cells using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of gamma-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 gamma-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 gamma-tubulin is required for microtubule nucleation throughout the mammalian cell cycle.