Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae.
Nucleation of microtubules in vitro by isolated spindle pole bodies of the yeast Saccharomyces cerevisiae.
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DOI：
10.1083/jcb.78.2.401
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发表时间：
1978-08
期刊：
影响因子：
--
通讯作者：
Borisy GG
中科院分区：
文献类型：
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作者：
Hyams JS;Borisy GG
采用Kleinschmidt单层分离技术，从酿酒酵母中分离到纺锤体。从细胞中制备的原生质体在空气-水界面上裂解。涂抹的制剂在网格上拾取，转移到实验测试溶液中，并准备用于整体安装的电子显微镜。用从猪脑组织中提纯的外源性微管蛋白，分离的SPBS在体外可以使微管组装成核。微管的生长是定向的，主要生长在SPB的核内面。用DNase、RNase或磷脂酶处理都不影响SPB的形态和微管启动能力，尽管这两种特性对胰酶都很敏感。对细胞周期不同阶段SPBS的分析表明，新复制的SPBS具有形成微管的能力。从指数级生长的细胞中分离出的SPBS启动了一组酵母纺锤体微管，其数量相当于体内看到的极到极微管的数量。然而，从处于稳定期的细胞中分离出来的SPBS在G1期停滞不前，在酵母纺锤体中形成了相当于总染色体和极至极小管的数量的微管。这可能意味着在G1期滞留细胞中，SPB与酵母染色质的微管附着位置有关。
Spindle pole bodies (SPBs) were isolated from the yeast Saccharomyces cerevisiae by an adaptation of the Kleinschmidt monolayer technique. Spheroplasts prepared from the cells were lysed on an air-water interface. Spread preparations were picked up on grids, transferred to experimental test solutions, and prepared for whole-mount electron microscopy. Using purified exogenous tubulin from porcine brain tissue, the isolated SPBs were shown to nucleate the assembly of microtubules in vitro. Microtubule growth was directional and primarily onto the intranuclear face of the SPB. Neither the morphology nor the microtubule-initiating capacity of the SPB was affected by treatment with the enzymes DNase, RNase, or phospholipase although both properties were sensitive to trypsin. Analysis of SPBs at various stages of the cell cycle showed that newly replicated SPBs had the capacity to nucleate microtubules. SPBs isolated from exponentially growing cells initiated a subset of the yeast spindle microtubules equivalent to the number of pole-to-pole microtubules seen in vivo. However, SPBs isolated from cells in stationary phase and therefore arrested in G1 nucleated a number of microtubules equal to the total chromosomal and pole-to-pole tubules in the yeast spindle. This may mean that in G1-arrested cells, the SPB is associated with microtubule attachment sites of the yeast chromatin.