Hyams J S, Borisy G G
J Cell Biol. 1978 Aug;78(2):401-14. doi: 10.1083/jcb.78.2.401.
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.
通过改良克莱因施密特单层技术，从酿酒酵母中分离出纺锤体极体（SPB）。将细胞制备的原生质球在气-水界面上裂解。将铺展的样品收集到网格上，转移到实验测试溶液中，并制备用于整装电子显微镜观察。使用从猪脑组织中纯化的外源微管蛋白，显示分离出的SPB在体外能促使微管组装。微管生长具有方向性，主要生长在SPB的核内面上。尽管SPB的形态和微管起始能力对胰蛋白酶敏感，但用脱氧核糖核酸酶（DNase）、核糖核酸酶（RNase）或磷脂酶处理均不影响其上述特性。对细胞周期不同阶段的SPB分析表明，新复制的SPB具有促使微管形成的能力。从指数生长期细胞中分离出的SPB起始的酵母纺锤体微管子集，其数量与体内观察到的极间微管数量相当。然而，从处于静止期因而停滞在G1期的细胞中分离出的SPB起始的微管数量，与酵母纺锤体中总的染色体微管和极间微管数量相等。这可能意味着，在停滞于G1期的细胞中，SPB与酵母染色质的微管附着位点相关。
