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.
|Number of pages||17|
|Journal||Membrane and Cell Biology|
|Publication status||Published - Nov 20 2000|
ASJC Scopus subject areas
- Cell Biology