Nature Genetics Advance Online Publication 1 Dynamics of the P53-mdm2 Feedback Loop in Individual Cells
Nature Genetics Advance Online Publication 1 Dynamics of the P53-mdm2 Feedback Loop in Individual Cells
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通讯作者：
G. Lahav;N. Rosenfeld;A. Sigal;N. Geva-Zatorsky;A. Levine;M. Elowitz;U. Alon
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作者：
G. Lahav;N. Rosenfeld;A. Sigal;N. Geva-Zatorsky;A. Levine;M. Elowitz;U. Alon
肿瘤抑制因子p53是研究最深入的蛋白质1-5之一，通常通过对细胞群进行平均的实验来研究，这可能掩盖了单个细胞中的动态行为。我们提出了一个系统，在单个活细胞中，p53及其负调节因子Mdm 2的动力学（参考文献1）。1，4-7）：该系统使用功能性p53-CFP和Mdm 2-YFP融合蛋白和延时荧光显微术。我们发现，p53在DNA损伤后以一系列离散脉冲的形式表达。基因相同的细胞有不同数量的脉冲：零，一，两个或更多。每个脉冲的平均高度和持续时间是固定的，不依赖于DNA损伤的量。然而，平均脉冲数随着DNA损伤而增加。这种方法可用于研究其他信号系统，并表明p53-Mdm 2反馈回路产生一个“数字”时钟，释放适时的p53量子，直到损伤修复或细胞死亡。生物体中常见的网络基序8是由一个转录臂和一个蛋白质相互作用臂组成的负反馈环（图1a）。在这里，我们选择了人类细胞中研究得最好的例子之一，其中肿瘤抑制因子p53转录激活Mdm 2，Mdm 2反过来靶向p53降解（图1b）1-5。在应激如DNA损伤后，Mdm 2活性降低a B c d e f g图1 p53-Mdm 2系统和荧光蛋白融合系统。(a)在哺乳动物10、27、果蝇28、酵母29和细菌30的不同系统中具有一个转录臂和一个蛋白质-蛋白质相互作用臂的负反馈环基序的实例。(b)p53-Mdm 2负反馈环：DNA损伤后，p53作为转录因子被激活，Mdm 2-p53相互作用降低1-7。(c)p53-CFP和Mdm 2-YFP构建体。(d)用GFP（和CFP）、p53和Mdm 2的抗体，在有或没有6小时锌诱导的情况下，在用p53、CFP或在pMT-I下表达的p53-CFP稳定转染的H1299细胞上进行免疫印迹。(e)用25 µg ml-1碘化丙啶染色，通过流式细胞术观察表达p53-CFP或CFP的H1299细胞（有或无48小时锌诱导）的细胞周期分布。Ap是亚G1区细胞的百分比，代表凋亡亚群。(f)5-戈伊照射后，在指定时间（h），p53和p53-CFP（抗p53抗体）以及Mdm 2和Mdm 2-YFP（抗Mdm 2抗体）的免疫印迹动力学..
The tumor suppressor p53, one of the most intensely investigated proteins 1–5 , is usually studied by experiments that are averaged over cell populations, potentially masking the dynamic behavior in individual cells. We present a system for following, in individual living cells, the dynamics of p53 and its negative regulator Mdm2 (refs. 1, 4–7): this system uses functional p53-CFP and Mdm2-YFP fusion proteins and time-lapse fluorescence microscopy. We found that p53 was expressed in a series of discrete pulses after DNA damage. Genetically identical cells had different numbers of pulses: zero, one, two or more. The mean height and duration of each pulse were fixed and did not depend on the amount of DNA damage. The mean number of pulses, however, increased with DNA damage. This approach can be used to study other signaling systems and suggests that the p53-Mdm2 feedback loop generates a 'digital' clock that releases well-timed quanta of p53 until damage is repaired or the cell dies. A common network motif 8 across organisms is a negative feedback loop composed of one transcription arm and one protein-interaction arm (Fig. 1a). Here we chose one of the best-studied examples in human cells, in which the tumor suppressor p53 transcriptionally activates Mdm2, which in turn targets p53 for degradation (Fig. 1b) 1–5. After stresses such as DNA damage, a decrease in the activity of Mdm2 a b c d e f g Figure 1 The p53-Mdm2 system and the fluorescent protein fusion system. (a) Examples of the negative feedback loop motif with one transcription arm and one protein-protein interaction arm in diverse systems in mammals 10,27 , fruit flies 28 , yeast 29 and bacteria 30. (b) The p53-Mdm2 negative feedback loop: after DNA damage, p53 is activated as a transcription factor and Mdm2-p53 interaction decreases 1–7. (c) p53-CFP and Mdm2-YFP constructs. (d) Immunoblots with antibodies to GFP (and CFP), p53 and Mdm2, with or without 6-h zinc induction, on H1299 cells stably transfected with p53, CFP or p53-CFP expressed under pMT-I. (e) Cell-cycle distribution by flow cytometry of H1299 cells expressing p53-CFP or CFP, with or without 48-h zinc induction, stained with 25 µg ml –1 of propidium iodide. Ap is the percentage of cells in the sub-G1 region, representing the apoptotic subpopulation. (f) Immunoblot kinetics of p53 and p53-CFP (antibody to p53) and Mdm2 and Mdm2-YFP (antibody to Mdm2) at the indicated times (in h) after 5-Gy …