γ-Tubulin distribution in the interphase and mitotic cell after stabilization or depolymerization of microtubules

Using indirect immunofluorescence and digital videomicroscopy we examined distribution of gamma-tubulin in mitotic and interphase HeLa cells after treatment with, mircotubule stabilizing (taxol) and microtubule depolymerizing (nocodazole) drugs. In interphase HeLa cells, affinity purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin specifically stained one dot or a pair of dots - centrioles. The contents of gamma-tubulin;in each of two centrioles within a cell were similar. The contents of gamma-tubulin in the mitotic poles were fourfold higher than in the interphase centrioles. In the interphase cells after nocodazole treatment (5 mu g/ml) the amount of gamma-tubulin in the centrosome slightly decreased, and after 24 h it was half of that in the control cells. In the mitotic cells 2 h after nocodazole treatment the amount of gamma-tubulin in mitotic poles decreased fourfold as compared to the control level. Under the action of nocodazole mitotic poles were stained unevenly - the intensivity of fluorescence in the center of the spot was weaker than at the periphery. After taxol treatment (10 mu g/ml), the amount of gamma-tubulin in the centrosomes of interphase cells decreased, then increased; after 24 h of incubation it doubled compared to the control level, and brightly stained spots appeared in cell cytoplasm along microtubule bundles. However, the content of gamma-tubulin per cell 24 h after taxol treatment didn't change. Two to 4 h after taxol treatment of the mitotic cells, the amount of gamma-tubuiin in the centrosome decreased twofold compared to the control level. In some cells, antibodies against gamma-tubulin stained up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. The results obtained give evidence that there are three pools of gamma-tubuiin: (1) constitutive gamma-tubulin that is permanently associated with the centrioles irrespective of the cell cycle stage and microtubule organizing capacity of the centrosome; (2) gamma-tubulin associated with the mitotic centrosome; its presence depends on both cell cycle stage (mitosis, interphase) and the presence of microtubules around the centrosome; (3) cytoplasmic gamma-tubulin that can associate with stable microtubules.