Cande W Z, McDonald K
J Cell Biol. 1986 Aug;103(2):593-604. doi: 10.1083/jcb.103.2.593.
We have developed a simple procedure for isolating mitotic spindles from the diatom Stephanopyxis turris and have shown that they undergo anaphase spindle elongation in vitro upon addition of ATP. The isolated central spindle is a barrel-shaped structure with a prominent zone of microtubule overlap. After ATP addition greater than 75% of the spindle population undergoes distinct structural rearrangements: the spindles on average are longer and the two half-spindles are separated by a distinct gap traversed by only a small number of microtubules, the phase-dense material in the overlap zone is gone, and the peripheral microtubule arrays have depolymerized. At the ultrastructural level, we examined serial cross-sections of spindles after 1-, 5-, and 10-min incubations in reactivation medium. Microtubule depolymerization distal to the poles is confirmed by the increased number of incomplete, i.e., c-microtubule profiles specifically located in the region of overlap. After 10 min we see areas of reduced microtubule number which correspond to the gaps seen in the light microscope and an overall reduction in the number of half-spindle microtubules to about one-third the original number. The changes in spindle structure are highly specific for ATP, are dose-dependent, and do not occur with nonhydrolyzable nucleotide analogues. Spindle elongation and gap formation are blocked by 10 microM vanadate, equimolar mixtures of ATP and AMPPNP, and by sulfhydryl reagents. This process is not affected by nocodazole, erythro-9-[3-(2-hydroxynonyl)]adenine, cytochalasin D, and phalloidin. In the presence of taxol, the extent of spindle elongation is increased; however, distinct gaps still form between the two half-spindles. These results show that the response of isolated spindles to ATP is a complex process consisting of several discrete steps including initiation events, spindle elongation mechanochemistry, controlled central spindle microtubule plus-end depolymerization, and loss of peripheral microtubules. They also show that the microtubule overlap zone is an important site of ATP action and suggest that spindle elongation in vitro is best explained by a mechanism of microtubule-microtubule sliding. Spindle elongation in vitro cannot be accounted for by cytoplasmic forces pulling on the poles or by microtubule polymerization.
我们开发了一种从硅藻Stephanopyxis turris中分离有丝分裂纺锤体的简单方法，并表明在添加ATP后，它们在体外会经历后期纺锤体伸长。分离出的中央纺锤体是一种桶状结构，具有明显的微管重叠区域。添加ATP后，超过75%的纺锤体群体发生明显的结构重排：纺锤体平均变长，两个半纺锤体由一个仅由少量微管穿过的明显间隙隔开，重叠区域的相致密物质消失，外周微管阵列解聚。在超微结构水平上，我们检查了在再激活培养基中孵育1分钟、5分钟和10分钟后纺锤体的连续横截面。极远端微管的解聚通过不完全微管（即c-微管轮廓）数量的增加得到证实，这些微管轮廓专门位于重叠区域。10分钟后，我们看到微管数量减少的区域，这与光学显微镜下看到的间隙相对应，并且半纺锤体微管的数量总体减少到原始数量的约三分之一。纺锤体结构的变化对ATP具有高度特异性，呈剂量依赖性，并且不会在不可水解的核苷酸类似物存在时发生。纺锤体伸长和间隙形成被10微摩尔钒酸盐、ATP和AMPPNP的等摩尔混合物以及巯基试剂阻断。这个过程不受诺考达唑、erythro-9-[3-(2-羟基壬基)]腺嘌呤、细胞松弛素D和鬼笔环肽的影响。在紫杉醇存在的情况下，纺锤体伸长的程度增加；然而，两个半纺锤体之间仍然形成明显的间隙。这些结果表明，分离的纺锤体对ATP的反应是一个复杂的过程，由几个离散的步骤组成，包括起始事件、纺锤体伸长的机械化学、受控的中央纺锤体微管正端解聚以及外周微管的丢失。它们还表明微管重叠区域是ATP作用的重要位点，并表明体外纺锤体伸长最好用微管-微管滑动机制来解释。体外纺锤体伸长不能用拉动两极的细胞质力或微管聚合来解释。
