Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI
Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI
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DOI：
10.1080/15384101.2018.1547001
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发表时间：
2018-11-17
期刊：
影响因子：
4.3
通讯作者：
Purvis, Jeremy E.
中科院分区：
文献类型：
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作者：
Grant, Gavin D.;Kedziora, Katarzyna M.;Purvis, Jeremy E.
细胞周期阶段转换是紧密协调的，以确保有效的细胞周期进程和基因组的稳定性。询问这些转变对于理解正常和病理性细胞增殖都很重要。通过量子化流行的Fucci记者相对于S相跃迁的动力学，我们发现他们的动力学实质上和可变地偏离了真实的S相界。为了增强对相变的检测，我们产生了一种新的报告器，其振荡直接与DNA复制相耦合，并将其与Fucci APC/C报告器相结合，创建了“PIP-Fucci”。PIP degron融合蛋白准确地标记了G1/S和S/G2的转变；仅在G1期显示出对大剂量DNA损伤的快速信号减弱；并区分了细胞类型特异性和DNA损伤来源依赖的停滞表型。我们为研究者选择合适的荧光细胞周期报告器和描述细胞周期转变的新的分析策略提供指导。
Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.