Gorbsky G J, Sammak P J, Borisy G G
J Cell Biol. 1987 Jan;104(1):9-18. doi: 10.1083/jcb.104.1.9.
During the movement of chromosomes in anaphase, microtubules that extend between the kinetochores and the poles shorten. We sought to determine where subunits are lost from these microtubules during their shortening. Prophase or prometaphase cells on coverslips were injected with fluoresceinated tubulin and allowed to progress through mitosis. Immediately after the onset of anaphase, a bar-shaped beam of laser light was used to mark a domain on the kinetochore fibers by photobleaching a band, approximately 1.0 micron wide, across the spindle. In different cells, spindles were photobleached at varying distances from the chromosomes. Cells were allowed to continue in anaphase until the chromosomes had further separated. They were then lysed, fixed, and prepared for double-label immunofluorescence with an antibody to fluorescein that does not bind appreciably to bleached fluorescein, and with an antibody to tubulin. Photobleached domains of microtubules appeared as bands of reduced fluorescence in the anti-fluorescein image. However, the anti-tubulin labeling revealed that microtubules were present and continuous through the photobleached domains. In all cases, the chromosomes approached and invaded the bleached domain while the bleached domain itself remained stationary with respect to the near pole. These results demonstrate that the chromosomes move along stationary kinetochore microtubules and that depolymerization of these microtubules during anaphase takes place at the kinetochore. In contrast to the generally accepted older view that chromosomes are passive objects pulled by "traction fibers," we suggest that the kinetochore is an active participant in generating the motive force that propels the chromosome to the pole.
在后期染色体移动过程中,连接着丝粒与纺锤体两极的微管会缩短。我们试图确定在这些微管缩短过程中,亚基是从何处丢失的。将带有荧光标记的微管蛋白注入盖玻片上处于前期或前中期的细胞中,使其进入有丝分裂过程。在后期开始后,立即用一束条形激光通过光漂白纺锤体上一条约1.0微米宽的带,来标记动粒纤维上的一个区域。在不同的细胞中,纺锤体在距染色体不同距离处进行光漂白。让细胞继续进行后期,直到染色体进一步分离。然后将细胞裂解、固定,并准备用一种与漂白后的荧光素无明显结合的抗荧光素抗体和一种抗微管蛋白抗体进行双重标记免疫荧光检测。在抗荧光素图像中,微管的光漂白区域呈现为荧光减弱的带。然而,抗微管蛋白标记显示微管存在且贯穿光漂白区域。在所有情况下,染色体靠近并侵入漂白区域,而漂白区域本身相对于近端纺锤体极保持静止。这些结果表明,染色体沿着静止的动粒微管移动,并且在后期这些微管的解聚发生在动粒处。与普遍接受的旧观点,即染色体是被“牵引纤维”拉动的被动物体相反,我们认为动粒是产生将染色体推向纺锤体极的动力的积极参与者。