King S M, Hyams J S, Luba A
J Cell Biol. 1982 Aug;94(2):341-9. doi: 10.1083/jcb.94.2.341.
Mitotic spindles were isolated from a cell division cycle mutant of the budding yeast Saccharomyces cerevisiae by the lysis of sphateroplasts on an air:buffer interface and were negatively stained with 1% gold thioglucose. Isolated spindles were incubated under conditions which promoted the sliding disintegration of parallel preparations of Tetrahymena axonemes, namely the addition of ATP to 20 microM. In no experiment was a corresponding change in microtubule organization of the spindle observed even when spindles were first pretreated with either 1-10 microgram/ml trypsin or 0.2-2% Triton X-100. During these experiments a number of spindles were isolated from cells that had passed through the imposed temperature block, and from the images obtained a detailed model of spindle formation and elongation has been constructed. Two sets of microtubules, one from each spindle pole body (SPB), completely interdigitate to form a continuous bundle, and a series of discontinuous microtubules are then nucleated by each SPB. As the spindle elongates, the number of microtubules continuous between the two SPBs decreases until, at a length of 4 micrometer, only one remains. The spindle, composed of only one microtubule, continues to elongate until it reaches the maximal nuclear dimension of 8 micrometer. The data obtained from negatively stained preparations have been verified in thin sections of wild-type cells. We suggest that, as in the later stages of mitosis only one microtubule is involved in the separation of the spindle poles, the microtubular spindle in S. cerevisiae is not a force-generating system but rather acts as a regulatory mechanism controlling the rate of separation.
缓冲液界面裂解球状体，从芽殖酵母酿酒酵母的细胞分裂周期突变体中分离出有丝分裂纺锤体，并用1%的硫代葡萄糖金进行负染色。将分离出的纺锤体在促进四膜虫轴丝平行制剂滑动解体的条件下孵育，即添加ATP至20微摩尔。即使纺锤体先用1 - 10微克/毫升胰蛋白酶或0.2 - 2% Triton X - 100预处理，在任何实验中都未观察到纺锤体微管组织的相应变化。在这些实验中，从经过设定温度阻滞的细胞中分离出了许多纺锤体，并根据获得的图像构建了纺锤体形成和伸长的详细模型。两组微管，每组来自一个纺锤体极体（SPB），完全相互交错形成一个连续的束，然后每个SPB形成一系列不连续的微管。随着纺锤体伸长，两个SPB之间连续的微管数量减少，直到长度达到4微米时，只剩下一根。由仅一根微管组成的纺锤体继续伸长，直到达到8微米的最大核尺寸。从负染色制剂获得的数据已在野生型细胞的薄切片中得到验证。我们认为，由于在有丝分裂后期只有一根微管参与纺锤体极的分离，酿酒酵母中的微管纺锤体不是一个产生力的系统，而是作为一种控制分离速率的调节机制。
