gamma-Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies

Cell Motil Cytoskeleton. 1996;33(1):38-51. doi: 10.1002/(SICI)1097-0169(1996)33:1<38::AID-CM5>3.0.CO;2-E.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibody Specificity
  • HeLa Cells
  • Humans
  • Macropodidae
  • Mice
  • Mice, Inbred BALB C
  • Microtubules / metabolism*
  • Mitosis*
  • Molecular Sequence Data
  • Nocodazole / pharmacology
  • Paclitaxel / pharmacology*
  • Tubulin / immunology
  • Tubulin / metabolism*
  • Tumor Cells, Cultured
  • Turkey

Substances

  • Antibodies, Monoclonal
  • Tubulin
  • Paclitaxel
  • Nocodazole