Hotta Takashi, Lee Yuh-Ru Julie, Higaki Takumi, Hashimoto Takashi, Liu Bo
Department of Plant Biology, College of Biological Sciences, University of California, Davis, Davis, CA, United States.
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States.
Front Cell Dev Biol. 2022 Jul 22;10:949345. doi: 10.3389/fcell.2022.949345. eCollection 2022.
Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in , the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other . The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles.
植物细胞通过利用微管负端定向的驱动蛋白-14马达形成无中心体纺锤体，其中微管（MTs）汇聚到两个结构未明确定义的极。迄今为止，尚不清楚在植物纺锤体中汇聚的双极MT阵列是否呈现统一的极，如果是，那么如何实现这一目标。在植物中的六类驱动蛋白-14马达中，驱动蛋白-14A马达ATK1（KatA）和ATK5在纺锤体形态发生中具有基本功能。为了了解这两种功能冗余的驱动蛋白-14A马达如何促进纺锤体组装，我们在相应的缺失突变体中表达了ATK1-GFP和ATK5-GFP融合蛋白，发现它们在功能上与其天然形式相当。尽管在核膜破裂之前ATK1是一种核蛋白而ATK5是细胞质蛋白，但在有丝分裂后期，这两种马达与纺锤体和成膜体MTs具有相似的均匀结合定位模式。我们发现，当细胞处于高渗条件下时，ATK1和ATK5会迅速集中到统一的极焦点。与此同时，纺锤体极变得完美聚焦，就好像存在着类似中心体的MT组织中心，ATK1和ATK5在那里高度富集，动粒纤维指向该中心。ATK1/ATK5突出的MTs与动粒纤维的MTs分离表明，这些马达使极间MTs发生易位。我们的蛋白质纯化和活细胞成像结果表明，ATK1和ATK5相互关联。应激诱导的纺锤体极汇聚还伴随着MT成核因子γ-微管蛋白向极的积累。这些结果得出结论，两种驱动蛋白-14A马达形成寡聚马达复合物，驱动MT向纺锤体极易位，以建立具有汇聚极的无中心体纺锤体。
