The Role of Nonmuscle Myosin II in Cytokinesis

在脊椎动物胞质分裂过程中，收缩环的收缩被认为是由反平行肌动蛋白丝的非肌肉肌球蛋白II（NM II）易位驱动的，类似于肌肉收缩。在这里，我们报告在体内，原位和体外观察，挑战这一假设。NM II-B是正常心肌细胞发育所必需的。在小鼠中消融NM II-B导致心肌细胞胞质分裂缺陷。令人惊讶的是，表达不能移位肌动蛋白丝并具有显著降低的MgATP酶活性的突变型NM II-B R709 C代替野生型NM II-B，成功地挽救了小鼠心脏中NM II-B消融的心肌细胞中的多核化。在培养的COS-7细胞中NM II-B的分级siRNA敲低揭示NM II的量限制收缩环收缩。延时分析表明，环收缩的速率和程度取决于NM II表达的水平。此外，运动受损的突变NM II（NM II-B R709 C、NM II-A N93 K和NM II-A R234 A）的表达恢复了NM II-B耗尽的COS-7细胞中的收缩环收缩，即使这些突变NM II不能移位肌动蛋白丝。然而，在表达野生型或突变型NM II的细胞中，收缩环收缩被blebbistatin阻断，blebistatin使NM II保持在弱结合（与肌动蛋白）状态。这些结果支持NM II在产生张力，但不易位肌动蛋白丝在收缩环收缩的作用。这一作用证实了体外瞬时动力学实验杆状病毒表达NM II蛋白使用停流分析。突变体NM II-B R709 C的机械化学性质显示出对肌动蛋白的极高亲和力，尽管失去了肌动蛋白易位。在加载条件下，突变NM II表现出长期的强肌动蛋白附着期间，一个单一的mechanoenzymatic周期跨越大部分时间的胞质分裂。这种长期的附着促进基本上所有NM II头肌动蛋白的同时结合，从而增加张力的产生和抵抗环和细胞皮质的扩张，但进一步防止肌动蛋白丝的易位。

During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin-filaments, similar to muscle contraction. Here we report in vivo, in situ and in vitro observations that challenge this hypothesis. NM II-B is essential for normal cardiac myocyte development. Ablation of NM II-B in mice resulted in defects in cardiac myocyte cytokinesis. Surprisingly, expression of mutant NM II-B R709C that cannot translocate actin filaments and has a substantially diminished MgATPase activity, in place of wild-type NM II-B, successfully rescues multinucleation in NM II-B ablated cardiomyocytes in mouse hearts. Graded siRNA knockdown of NM II-B in cultured COS-7 cells reveals that the amount of NM II limits contractile ring constriction. Time-lapse analyses show that both the rate and extent of ring constriction depends on the level of NM II expression. In addition expression of motor-impaired mutant NM IIs (NM II-B R709C, NM II-A N93K and NM II-A R234A) restores contractile ring constriction in COS-7 cells depleted of NM II-B, even though these mutant NM IIs are incapable of translocating actin-filaments. However contractile ring constriction is blocked by blebbistatin which keeps NM II in the weakly bound (to actin) state, in cells expressing either wild-type or mutant NM IIs. These results support a role for NM II in generating tension but not translocating actin-filaments during contractile ring constriction. This role is substantiated by in vitro transient kinetic experiments with baculovirus-expressed NM II proteins using stopped-flow analyses. The mechanochemical properties of mutant NM II-B R709C show extremely high affinity for actin, despite loss of actin translocation. Under loaded conditions, mutant NM II exhibits prolonged strong actin attachment during which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of essentially all NM II heads to actin, thereby increasing tension generation and resisting expansion of the ring and cell cortex, but further preventing translocation of actin-filaments.