Nicklas R B, Kubai D F, Hays T S
J Cell Biol. 1982 Oct;95(1):91-104. doi: 10.1083/jcb.95.1.91.
Micromanipulation of living grasshopper spermatocytes in anaphase has been combined with electron microscopy to reveal otherwise obscure features of spindle organization. A chromosome is pushed laterally outside the spindle and stretched, and the cell is fixed with a novel, agar-treated glutaraldehyde solution. Two- and three-dimensional reconstructions from serial sections of seven cells show that kinetochore microtubules of the manipulated chromosome are shifted outside the confusing thicket of spindle microtubules and mechanical associations among microtubules are revealed by bent or shifted microtubules. These are the chief results: (a) The disposition of microtubules invariably is consistent with a skeletal role for spindle microtubules. (b) The kinetochore microtubule bundle is composed of short and long microtubules, with weak but recognizable mechanical associations among them. Some kinetochore microtubules are more tightly linked to one other microtubule within the bundle. (c) Microtubules of the kinetochore microtubule bundle are firmly connected to other spindle microtubules only near the pole, although some nonkinetochore microtubules of uncertain significance enter the bundle nearer to the kinetochore. (d) The kinetochore microtubules of adjacent chromosomes are mechanically linked, which provides an explanation for interdependent chromosome movement in "hinge anaphases." In the region of the spindle open to analysis after chromosome micromanipulation, microtubules may be linked mechanically by embedment in a gel, rather than by dynein or other specific, cross-bridging molecules.
对处于后期的活蝗虫精母细胞进行显微操作,并结合电子显微镜技术,以揭示纺锤体组织中其他难以察觉的特征。将一条染色体侧向推至纺锤体外部并拉伸,然后用一种新型的经琼脂处理的戊二醛溶液固定细胞。对七个细胞的连续切片进行二维和三维重建显示,被操作染色体的动粒微管移至纺锤体微管的复杂丛之外,并且微管之间的机械关联通过弯曲或移位的微管得以揭示。主要结果如下:(a) 微管的排列始终与纺锤体微管的骨架作用一致。(b) 动粒微管束由短微管和长微管组成,它们之间存在微弱但可识别的机械关联。一些动粒微管在束内与另一微管的连接更为紧密。(c) 动粒微管束的微管仅在靠近极的位置与其他纺锤体微管牢固连接,尽管一些意义不明的非动粒微管在更靠近动粒的位置进入该束。(d) 相邻染色体的动粒微管存在机械连接,这为“铰链后期”中染色体的相互依赖运动提供了解释。在染色体显微操作后可供分析的纺锤体区域,微管可能通过嵌入凝胶而在机械上相互连接,而非通过动力蛋白或其他特定的交叉桥接分子。