Live-cell imaging provides direct evidence for a threshold in CDK activity at the G2/M transition
Live-cell imaging provides direct evidence for a threshold in CDK activity at the G2/M transition
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活细胞成像为 G2/M 转变时 CDK 活性阈值提供了直接证据
DOI：
10.1101/2023.03.26.534249
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Aoki Kazuhiro
中科院分区：
文献类型：
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作者：
Sugiyama Hironori;Goto Yuhei;Kondo Yohei;Coudreuse Damien;Aoki Kazuhiro
细胞周期蛋白依赖性激酶(CDK)在决定细胞周期时相的时间顺序中起着至关重要的作用。然而，尽管对CDK调控因子的研究取得了重大进展，但它们如何在单细胞水平上协同影响CDK活性，以及CDK如何控制细胞周期事件的时间顺序仍然是个未知数。这可能是由于缺乏监测活细胞中CDK活动的工具。在这里，我们利用一种新开发的基于Förster共振能量转移(FRET)的CDK活性生物传感器Eevee-spCDK来阐明分裂酵母和哺乳动物细胞中CDK活性的动态变化。利用这个系统，我们揭示了CDK活性在营养生长中的分布。庞贝壳。因此，我们检测到S阶段的瞬时增加，然后在G2阶段逐渐增加。CDK活性在M期早期达到最大值，在有丝分裂退出时迅速下降。在G2期，正如数学研究预测的那样，CDK活性呈现出一个双相模式，即在G2/M相变之前，早期缓慢上升，晚期快速上升。值得注意的是，尽管cdk活性与细胞周期蛋白水平并不一定相关，但我们发现，在几个突变背景中，cdk活性在有丝分裂开始时收敛到相同的水平，包括pom1Δ细胞和Weet 1orcdc25过表达细胞。这些数据首次提供了细胞在CDK活性达到高阈值时进入M期的直接证据，这与分裂酵母细胞周期进程的定量模型一致。
Cyclin-dependent kinase (CDK) plays an essential role in determining the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK, it remains elusive how they coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. This could be due to the lack of tools to monitor CDK activity in living cells. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by using a newly developed CDK activity biosensor, Eevee-spCDK, based on Förster Resonance Energy Transfer (FRET). Taking advantage of this system, we unravel the profile of CDK activity in vegetatively growingS. pombecells. Thus, we detect a transient increase in S phase followed by a gradual increment during G2 phase. CDK activity then reaches its maximum in early M phase and rapidly decreases at mitotic exit. During G2 phase, CDK activity exhibits a biphasic pattern,i.e., an early slow increase and a late fast rise prior to the G2/M phase transition, as predicted from mathematical studies. Remarkably, although CDK activity does not necessarily correlate with cyclin levels, we find that it converges to the same level around mitotic onset in several mutant backgrounds, includingpom1Δcells andwee1orcdc25overexpressing cells. These data provide the first direct evidence that cells enter M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.