Institute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia.
Laboratory of Bioengineering, Novosibirsk State Agrarian University, 630039 Novosibirsk, Russia.
Cells. 2022 Jul 6;11(14):2127. doi: 10.3390/cells11142127.
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: // (), (), (), (), (), (), (), and (). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.
中心体含有细胞利用三种主要类型的微管（MTs）组装纺锤体：由中心体产生的 MTs、靠近染色体/动粒的 MTs 和由 augmin 依赖途径在纺锤体内产生的 MTs。哺乳动物和 细胞缺乏中心体，在动粒处产生 MT，并最终形成功能性双极纺锤体。然而，动粒驱动的 MT 形成的机制还不太清楚。阐明这些机制的一种方法是分析 MT 解聚后纺锤体的重新组装。在这里，我们使用基于 RNA 干扰（RNAi）的反向遗传学方法来剖析秋水仙素诱导 MT 解聚后动粒驱动的 MT 再生（KDMTR）的过程。这种 MT 解聚程序允许清楚地评估 KDMTR，因为秋水仙素破坏了中心体驱动的 MT 再生，但不影响 KDMTR。我们检查了正常 S2 细胞和经 RNAi 处理的 S2 细胞中的 KDMTR，这些 RNAi 针对参与有丝分裂纺锤体组装的保守基因：//（）、（）、（）、（）、（）、（）、（）和（）。RNAi 介导的 Mast/Orbit、Mei-38、Mars、Dgt6 和 Eb1 的消耗导致 KDMTR 显著延迟，而 Patronin 的缺失对该过程的影响较轻。相比之下，Asp 或 Klp10A 的缺乏增加了 KDMTR 的速度。这些结果与 GFP 标记蛋白（Mast/Orbit、Mei-38、Mars、Eb1、Patronin 和 Asp）在 KDMTR 期间的定位分析相结合，提出了一个依赖动粒的纺锤体重新组装的模型。我们提出，动粒捕获其附近产生的 MT 的正极，并通过 Mast/Orbit 的作用使这些 MT 在动粒处伸长。Asp 蛋白从 KDMTR 开始就结合 MT 的负极，防止过度和无序的 MT 再生。Mei-38、Mars、Dgt6、Eb1 和 Patronin 正向调节聚合、束集和重新生长的 MT 的稳定，直到形成双极纺锤体。
