Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4509-14. doi: 10.1073/pnas.1116268109. Epub 2012 Mar 5.
During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin filaments. Here we report in situ, in vitro, and in vivo observations that challenge this hypothesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not required for cytokinesis. Blebbistatin inhibition of cytokinesis indicates the importance of myosin strongly binding to actin and exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the load-dependent mechanochemical properties of mutant NM II support efficient tension maintenance despite the inability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of NM II heads to actin, thereby increasing tension and resisting expansion of the ring. The detachment of mutant NM II heads from actin is enhanced by assisting loads, which prevent mutant NM II from hampering furrow ingression during cytokinesis. In the 3D context of mouse hearts, mutant NM II-B R709C that cannot translocate actin filaments can rescue multinucleation in NM II-B ablated cardiomyocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and cross-link actin filaments and to exert tension on actin during contractile ring constriction.
在脊椎动物胞质分裂过程中，收缩环的收缩被认为是由非肌肉肌球蛋白 II（NM II）的反平行肌动蛋白丝的易位驱动的。在这里，我们报告了原位、体外和体内观察结果，这些结果对这一假设提出了挑战。在培养的 COS-7 细胞中对 NM II 进行分级敲低，揭示了 NM II 的数量限制了环的收缩。用运动受损的 NM II 突变体恢复收缩率表明，NM II 易位肌动蛋白的能力对于胞质分裂不是必需的。胞质分裂的 blebbistatin 抑制表明，肌球蛋白在胞质分裂过程中强烈结合肌动蛋白并施加张力是很重要的。瞬态动力学实验证实了这一作用，表明突变 NM II 的负载依赖性机械化学特性支持有效的张力维持，尽管不能易位肌动蛋白。在加载条件下，突变 NM II 表现出延长的肌动蛋白附着，其中一个机械酶循环跨越了胞质分裂的大部分时间。这种延长的附着促进了 NM II 头部与肌动蛋白的同时结合，从而增加了张力并抵抗了环的扩张。通过辅助负载增强了突变 NM II 头部与肌动蛋白的分离，这阻止了突变 NM II 在胞质分裂过程中阻碍沟的侵入。在小鼠心脏的 3D 环境中，不能易位肌动蛋白丝的突变 NM II-B R709C 可以挽救 NM II-B 缺失的心肌细胞中的多核化。我们提出，NM II 在脊椎动物细胞胞质分裂中的主要作用是结合和交联肌动蛋白丝，并在收缩环收缩时对肌动蛋白施加张力。
