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 microtubule organizing centres and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans. 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.
Joshi
H C
HC
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
Palacios
M J
MJ
McNamara
L
L
Cleveland
D W
DW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002875:Chromosomes / Q000378:metabolism; D003080:Cold Temperature; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
D009687:Nuclear Proteins; D010446:Peptide Fragments; D014404:Tubulin; D015739:Nocodazole