gamma-Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies.
Monoclonal antibodies were prepared against conserved synthetic peptide from the C-terminus of the gamma-tubulin and their specificity was confirmed by immunoblotting, competitive enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The antibodies decorated interphase centrosomes as well as half-spindles and midbodies in mitotic cells of various origin. The prepared antibodies were used to study the gamma-tubulin distribution in nocodazole and taxol-treated cells. In the cells recovering from the nocodazole treatment, gamma-tubulin was found in centers of all microtubule asters. Examination of relative location of gamma-tubulin and microtubule asters in taxol-treated mitotic cells 3T3, HeLa and PtK2 revealed that the number of taxol-induced microtubule asters exceeded the number of gamma-tubulin-positive spots. The gamma-tubulin was often found in the periphery of microtubule asters. Centrosomal phosphoprotein epitope detected by MPM-2 antibody colocalized with gamma-tubulin in taxol-treated mitotic cells. The presented data suggest that taxol-induced microtubule asters are in vivo nucleated independently of gamma-tubulin, and other minus-end nucleator(s) are necessary for formation of such asters. Alternatively, gamma-tubulin is present in subthreshold amounts undetectable by immunofluorescence.
Nováková
M
M
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic.
Dráberová
E
E
Schürmann
W
W
Czihak
G
G
Viklický
V
V
Dr-aber
P
P
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D006367:HeLa Cells; D006801:Humans; D007614:Macropodidae; D051379:Mice; D008807:Mice, Inbred BALB C; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology*; D014404:Tubulin / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014421:Turkey
D000911:Antibodies, Monoclonal; D014404:Tubulin; D017239:Paclitaxel; D015739:Nocodazole