Laboratory for Cell Function Dynamics, BSI, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; King's College London Centre for Stem Cells and Regenerative Medicine, 28th Floor, Tower Wing, Guy's Campus, Great Maze Pond, London SE1 9RT, UK.
Mol Cell. 2017 Nov 2;68(3):626-640.e5. doi: 10.1016/j.molcel.2017.10.001. Epub 2017 Oct 26.
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-mediated ubiquitylation alone or in combination with SCF-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.
真核细胞在细胞周期的间期大部分时间都处于间期。为了了解间期发生的丰富多样的代谢和基因组调控，需要在原位标记精确的相位边界。在这里，我们报告了两种遗传编码荧光传感器 Fucci(CA)和 Fucci(SCA)的特性，它们能够实时监测间期和细胞周期生物学。我们对原始 Fucci 系统中的基于 Cdt1 的传感器进行了重新设计，使其仅响应 S 期特异性的 CUL4 介导的泛素化或与 SCF 介导的泛素化结合。在培养细胞中，Fucci(CA)产生了 G1、S 和 G2 之间明显的三重颜色区分，而 Fucci(SCA)则允许对 G1 和 S/G2 进行双色读数。Fucci(CA)的应用包括跟踪快速分裂的小鼠胚胎干细胞的短暂 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)是定量研究间期细胞周期调控的重要工具。
