Mitchison T, Evans L, Schulze E, Kirschner M
Cell. 1986 May 23;45(4):515-27. doi: 10.1016/0092-8674(86)90283-7.
We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.
我们已将生物素化微管蛋白显微注射到有丝分裂的成纤维细胞中，以确定纺锤体中新生亚基并入微管（MTs）的位点。使用抗生物素抗体，随后是与胶体金偶联的二抗，在电子显微镜下观察标记的亚基。星体微管通过现有微管的延长以及从中心体的新成核作用，非常迅速地并入标记的亚基。在中期，动粒微管以较慢的速率在动粒处逐渐并入亚基，这表明动粒纤维中的亚基向极缓慢流动。当检查中期注射的细胞在后期的情况时，相当一部分动粒微管未被标记，这表明在动粒处伴随着染色体向极移动发生了解聚。中期和后期动粒处相反流动的存在表明，两种独立的力量负责染色体的汇聚和后期移动。
