The kinetics of G2 and M transitions regulated by B cyclins.
The kinetics of G2 and M transitions regulated by B cyclins.
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DOI：
10.1371/journal.pone.0080861
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Jacobberger JW
中科院分区：
文献类型：
--
作者：
Huang Y;Sramkoski RM;Jacobberger JW
B细胞周期蛋白调节G2-M转换。由于人类体细胞在通过RNA干扰(RNAi)降低细胞周期蛋白B1(CycB1)或细胞周期蛋白B2(CycB2)后继续进行细胞周期，而且由于cycB2基因敲除小鼠是存活的，因此两个基因的存在应该是一种优化。为了探索这一想法，我们用诱导的细胞周期蛋白B1或B2-绿色荧光蛋白建立了抗™的HeLa BD RNAi去分化细胞系。用RNAi和/或多西环素(Dox)和溴脱氧尿苷处理培养物。我们测量了G2和M通过时间和4C细胞积聚。在没有异位B细胞周期蛋白表达的情况下，任何一种细胞周期蛋白的敲除(Kd)增加了G2转运。CycB1kd可增加M转运，而CycB2缺失可使M转运减少。这种新的差异进一步得到了时间推移显微镜的支持。这表明cycB2调节有丝分裂的时间，我们推测这是通过调节高尔基体检查点。在内源性细胞周期蛋白存在的情况下，活化的B细胞周期蛋白-EGFP的表达不影响G2或M期时间。如前所述，B细胞周期蛋白联合耗竭诱导了G2期细胞停滞。在单次kd实验中，B期细胞周期蛋白-绿色荧光蛋白的表达完全挽救了内源性细胞周期蛋白的下调，而B细胞周期蛋白-绿色荧光蛋白的表达则完全挽救了内源性细胞周期蛋白的共同耗竭。大多数救援发生在外源细胞周期蛋白表达水平相对较低的情况下。因此，在本实验环境中，cycB1和cycB2可以互换，以促进G2和M转换。周期蛋白B1被认为是哺乳动物体细胞周期所必需的，而周期蛋白B2被认为是必不可少的。然而，在双基因敲除实验中，Cyclin B1或Cyclin B2的残留水平不足以促进成功的有丝分裂，但在可分配的Cyclin B2存在的情况下，残余水平足以促进有丝分裂。我们讨论了一个简单的模型，该模型可以解释大多数数据，如果细胞周期蛋白B1是必要的。
B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary.
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