Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle
Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle
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DOI：
10.1016/j.molcel.2017.10.001
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发表时间：
2017-11-02
期刊：
影响因子：
16
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
--
作者：
Sakaue-Sawano, Asako;Yo, Masahiro;Miyawaki, Atsushi
真核细胞的大部分生命都处于细胞周期的间期。了解发生在间期的代谢和基因组调控的丰富多样性，需要在原位划定精确的相边界。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。我们从原始的Fucci系统中重新设计了基于Cdt 1的传感器，以响应S期特异性CUL 4 Ddb 1介导的泛素化，单独或与SCFSkp 2介导的泛素化结合。在培养的细胞中，Fucci（CA）产生了G1，S和G2的三种明显的颜色分离，而Fucci（SCA）允许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短暂G1期，并确定S期紫外线辐射损伤的窗口。这些结果表明Fucci（CA）是定量研究间期细胞周期调节的重要工具。
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 realtime 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 SCFSkp2 - 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 UVirradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.