Tetrad Graduate Program, University of California, San Francisco, San Francisco, United States.
Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, San Francisco, United States.
Elife. 2023 Jul 3;12:e85208. doi: 10.7554/eLife.85208.
At each cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the pole's contribution to mammalian spindle length, dynamics, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.
在每个细胞分裂过程中,纳米级组件自组织形成微米级纺锤体。在哺乳动物纺锤体中,称为动粒纤维的微管束附着在染色体上,并聚焦到纺锤体两极。尽管有证据表明两极可以设定纺锤体的长度,但它们的作用仍知之甚少。事实上,许多物种没有纺锤体两极。在这里,我们通过抑制动力蛋白来产生纺锤体,从而探测两极对哺乳动物纺锤体长度、动态和功能的贡献,这些纺锤体的动粒纤维没有聚焦到两极,但保持着中期的稳态长度。我们发现,未聚焦的动粒纤维的平均长度与对照无明显差异,但长度分布更宽,姐妹和邻居之间的长度协调性降低。此外,我们还表明,未聚焦的动粒纤维与对照一样,如果被药物处理或激光消融急性缩短,可以恢复到其稳态长度:它们通过调整末端动力学来恢复长度,尽管由于基础动力学降低而速度较慢。因此,动粒纤维的动力学受到其长度的调节,而不仅仅是两极聚焦力的调节。最后,我们表明,具有未聚焦动粒纤维的纺锤体可以分离染色体,但不能正确分离。我们提出,哺乳动物纺锤体的长度是由单个动粒纤维局部产生的,而纺锤体两极则在空间和时间上全局协调动粒纤维。