γ-Tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

I. A. Vorobjev, R. E. Uzbekov, Yu A. Komarova, I. B. Alieva

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Indirect immunofluorescence and digital videomicroscopy were used to study γ-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against γ-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The γ-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of γ-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 μg/ml) on interphase cells resulted in lowering the amount of γ-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the γ-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 μg/ml), the γ-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular γ-tubulin did not change. Treatment with taxol for 2-4 h halved the γ-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against γ-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three γ-tubulin pools is suggested: (1) constitutive γ-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) γ-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic γ-tubulin that can bind to stable microtubules.

Original languageEnglish
Pages (from-to)219-235
Number of pages17
JournalMembrane and Cell Biology
Volume14
Issue number2
Publication statusPublished - Nov 20 2000

ASJC Scopus subject areas

  • Cell Biology

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