Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4^Ddb1-mediated ubiquitylation alone or in combination with SCF^Skp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.

真核细胞的大部分生命都在细胞周期的中期进行。要了解在相间发生的代谢和基因组调控的丰富多样性，就需要对精确的相界进行原位划分。在这里，我们报告两个遗传编码的荧光传感器Fucci（CA）和Fucci（SCA）的特性，它们可以对相间和细胞周期生物学进行实时监控。我们对原始Fucci系统中基于Cdt1的传感器进行了重新设计，以单独或与SCF ^Skp2结合对S期特异性CUL4 ^Ddb1介导的泛素化反应介导的泛素化。在培养的细胞中，Fucci（CA）产生了G1，S和G2的清晰的三重色差分离，而Fucci（SCA）允许了G1和S / G2的双色读数。Fucci（CA）的应用包括跟踪快速分裂的小鼠胚胎干细胞的瞬时G1期，并确定S期中紫外线照射损伤的窗口。这些结果表明Fucci（CA）是定量研究相间细胞周期调控的重要工具。