Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.
Visualizing and Quantifying Intracellular Behavior and Abundance of the Core Circadian Clock Protein PERIOD2.
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DOI：
10.1016/j.cub.2016.05.018
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发表时间：
2016-07-25
期刊：
影响因子：
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通讯作者：
Loudon AS
中科院分区：
文献类型：
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作者：
Smyllie NJ;Pilorz V;Boyd J;Meng QJ;Saer B;Chesham JE;Maywood ES;Krogager TP;Spiller DG;Boot-Handford R;White MR;Hastings MH;Loudon AS
转录-翻译反馈环（TTFLs）是生物钟的保守分子基序。哺乳动物的主要时钟是下丘脑的视交叉上核（SCN）。在SCN神经元中，核心时钟基因Period（Per）和Cryptochrome（Cry）的蛋白产物进入核后对其的自动调节反馈是昼夜节律振荡的基础。在果蝇的时钟神经元中，dPer进入细胞核的运动受到一个昼夜节律门的影响，该门在TTFL中产生延迟，并且这种延迟被认为是振荡的关键。果蝇生物钟的分析强烈地影响了哺乳动物生物钟的模型，这些模型通常推断哺乳动物生物钟蛋白的复杂时空细胞内行为。然而，没有内源性昼夜节律蛋白的细胞内行为的直接测量来支持这一点：动态分析受到限制，并且通常没有昼夜节律维度。因此，我们产生了表达天然PER 2蛋白的荧光融合物（PER 2：：VENUS）的敲入小鼠用于活体成像。PER 2：：VENUS重现了野生型PER 2的昼夜节律功能，重要的是，PER 2：：VENUS的行为与果蝇模型相反：它不表现出细胞核进入的昼夜节律门控。使用荧光成像的PER 2：：VENUS，我们获得了第一个措施的流动性，分子浓度和本地化的内源性昼夜节律蛋白在个别哺乳动物细胞，我们显示了如何流动性和核转位的PER 2调节酪蛋白激酶。这些结果为哺乳动物生物钟的细胞机制提供了新的定性和定量的见解。报告小鼠用于哺乳动物时钟蛋白PER 2的实时荧光成像与果蝇相反，PER 2的定位不受昼夜节律门控的影响。天然PER 2的昼夜节律丰度、移动性和细胞内动力学被定量。使用荧光报告小鼠对PER 2的昼夜动态进行成像，PER 2是视交叉上核和成纤维细胞中生物钟的关键成分。它们揭示了小鼠和苍蝇时钟细胞机制的显著差异，并提供了定量数据来支持对哺乳动物时钟当前模型的重新评估。
Transcriptional-translational feedback loops (TTFLs) are a conserved molecular motif of circadian clocks. The principal clock in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus. In SCN neurons, auto-regulatory feedback on core clock genes Period (Per) and Cryptochrome (Cry) following nuclear entry of their protein products is the basis of circadian oscillation. In Drosophila clock neurons, the movement of dPer into the nucleus is subject to a circadian gate that generates a delay in the TTFL, and this delay is thought to be critical for oscillation. Analysis of the Drosophila clock has strongly influenced models of the mammalian clock, and such models typically infer complex spatiotemporal, intracellular behaviors of mammalian clock proteins. There are, however, no direct measures of the intracellular behavior of endogenous circadian proteins to support this: dynamic analyses have been limited and often have no circadian dimension. We therefore generated a knockin mouse expressing a fluorescent fusion of native PER2 protein (PER2::VENUS) for live imaging. PER2::VENUS recapitulates the circadian functions of wild-type PER2 and, importantly, the behavior of PER2::VENUS runs counter to the Drosophila model: it does not exhibit circadian gating of nuclear entry. Using fluorescent imaging of PER2::VENUS, we acquired the first measures of mobility, molecular concentration, and localization of an endogenous circadian protein in individual mammalian cells, and we showed how the mobility and nuclear translocation of PER2 are regulated by casein kinase. These results provide new qualitative and quantitative insights into the cellular mechanism of the mammalian circadian clock. Reporter mouse is used for real-time fluorescent imaging of mammalian clock protein PER2 In contrast to Drosophila, localization of PER2 is not subject to circadian gating Circadian abundance, mobility, and intracellular dynamics of native PER2 are quantified Casein kinase1 controls nucleocytoplasmic mobility of PER2 alongside circadian period Smyllie et al. use a fluorescent reporter mouse to image the circadian dynamics of PER2, a key component of the circadian clock in the suprachiasmatic nucleus and fibroblasts. They reveal marked divergence of the mechanisms of mouse and fly clock cells, and they provide quantitative data to support reappraisal of current models of the mammalian clock.