γ-tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies

Abstract

Monoclonal antibodies were prepared against conserved synthetic peptide from the C-terminus of the γ-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 γ-tubulin distribution in nocodazole and taxol-treated cells. In the cells recovering from the nocodazole treatment, γ-tubulin was found in centers of all microtubule asters. Examination of relative location of γ-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 γ-tubulin-positive spots. The γ-tubulin was often found in the periphery of microtubule asters. Centrosomal phosphoprotein epitope detected by MPM-2 antibody colocalized with γ-tubulin in taxol-treated mitotic cells. The presented data suggest that taxol-induced microtubule asters are in vivo nucleated independently of γ-tubulin, and other minus-end nucleator(s) are necessary for formation of such asters. Alternatively, γ-tubulin is present in subthreshold amounts undetectable by immunofluorescence.