Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.

细胞周期蛋白依赖性激酶 (CDK) 决定细胞周期阶段的时间顺序。然而，尽管在群体水平上研究 CDK 调节剂和 CDK 底物磷酸化模式方面取得了重大进展，但 CDK 调节剂如何在单细胞水平上协调影响 CDK 活性以及 CDK 如何控制细胞周期事件的时间顺序仍然难以捉摸。在这里，我们通过开发 CDK 活性生物传感器 Eevee-spCDK阐明了裂殖酵母和哺乳动物细胞中 CDK 活性的动态。我们发现，尽管 CDK 活性不一定与细胞周期蛋白水平相关，但在几种突变背景（包括pom1Δ细胞和wee1或cdc25过表达细胞）中，它在有丝分裂开始时收敛到相同水平。这些数据提供了直接证据，表明当 CDK 活性达到高阈值时细胞进入 M 期，与裂殖酵母细胞周期进展的定量模型一致。