Byers B, Shriver K, Goetsch L
J Cell Sci. 1978 Apr;30:331-52. doi: 10.1242/jcs.30.1.331.
The spindle poles of the budding yeast, Saccharomyces cerevisiae, have been removed from mitotic and meiotic cells by osmotic lysis of spheroplasts. The spindle pole bodies (SPBs)--diskoidal structures also termed 'spindle plaques'--have been analysed for their ability to potentiate the polymerization of microtubules in vitro. Free SPBs were completely deprived of any detectable native microtubules by incubation in the absence of added tubulin and were then challenged with chick neurotubulin, which had been rendered partially defective in self-initiation of repolymerization. Electron microscopy revealed that these SPBs served as foci for the initiation of microtubule polymerization in vitro. Because the attached microtubules elongated linearly with time but did not increase in numbers after the first stage of the reaction, it is apparent that there are a limited number of sites for initiation. The initiating potential of the SPBs was found to be inhibited by enzymic hydrolysis of protein but not of DNA. The microtubule end proximal to the site of initiation on the SPB is distinguished by a 'closed' appearance because of a terminal component which is continuous with the microtubule wall, whereas the distal end has the 'open' appearance characteristic of freely repolymerized neurotubules. SPBs which were partially purified on sucrose gradients retained their ability to initiate the assembly of microtubules with the same structural differentiation of their ends. The occurrence of closed proximal ends on native yeast microtubules suggests that closed ends may play a role in the initiation of microtubule polymerization in vivo, as well as in vitro.
通过原生质球的渗透裂解，已从有丝分裂和减数分裂的细胞中移除了出芽酵母酿酒酵母的纺锤极。对纺锤极体（SPB）——也称为“纺锤体斑”的盘状结构——在体外增强微管聚合的能力进行了分析。通过在不添加微管蛋白的情况下孵育，游离的SPB完全去除了任何可检测到的天然微管，然后用在再聚合的自我起始方面部分有缺陷的鸡神经微管蛋白进行挑战。电子显微镜显示，这些SPB在体外作为微管聚合起始的位点。由于附着的微管随时间线性伸长，但在反应的第一阶段后数量没有增加，显然起始位点的数量是有限的。发现SPB的起始潜力受到蛋白质而非DNA的酶促水解的抑制。靠近SPB上起始位点的微管末端由于与微管壁连续的末端成分而呈现“封闭”外观，而远端具有自由再聚合的神经微管特有的“开放”外观。在蔗糖梯度上部分纯化的SPB保留了其起始微管组装的能力，其末端具有相同的结构差异。天然酵母微管上封闭近端的出现表明，封闭末端可能在体内以及体外微管聚合的起始中发挥作用。
