Vorobjev I A, Uzbekov R E, Alieva I B
Belozersky Institute of Physicochemical Biology, Moscow State University, Russia.
Membr Cell Biol. 2000;14(2):219-35.
Indirect immunofluorescence and digital videomicroscopy were used to study gamma-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 gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-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 gamma-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 microg/ml), the gamma-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 gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.
采用间接免疫荧光和数字视频显微镜技术，研究了γ-微管蛋白在正常有丝分裂期和间期HeLa细胞中的分布情况，以及这些细胞在用微管稳定药物（紫杉醇）和解聚药物（诺考达唑）处理后的情况。在间期HeLa细胞中，针对γ-微管蛋白的亲和纯化抗体和针对乙酰化微管蛋白的单克隆抗体可标记一两个相邻的点，即中心粒。来自同一细胞的两个中心粒中的γ-微管蛋白含量差异不显著。有丝分裂极中的γ-微管蛋白含量是间期中心粒的四倍。诺考达唑（5微克/毫升）对间期细胞的作用导致中心体中γ-微管蛋白含量降低，24小时后减少了一半。用诺考达唑处理2小时导致有丝分裂极中γ-微管蛋白含量减少四倍。此外，有丝分裂极的染色不均匀，中心的荧光强度低于周边。在用紫杉醇（10微克/毫升）处理后，间期细胞中心体中的γ-微管蛋白含量先降低，然后升高，24小时后与对照相比增加了一倍。在后一种情况下，沿着微管束在细胞质中出现明亮的点。然而，用紫杉醇处理24小时后，细胞内γ-微管蛋白的总量没有变化。与正常有丝分裂相比，用紫杉醇处理2至4小时使中心体中γ-微管蛋白含量减半。在一些细胞中，针对γ-微管蛋白的抗体显示多达四个微管汇聚焦点。其他众多的微管汇聚焦点未被染色。因此，提示至少存在三个γ-微管蛋白池：（1）组成型γ-微管蛋白，无论细胞周期阶段如何，都与中心粒永久结合，并且具有作为微管组织中心的能力；（2）仅在有丝分裂期间与中心体不稳定结合的γ-微管蛋白；（3）可与稳定微管结合的细胞质γ-微管蛋白。
