Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons.
Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons.
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DOI：
10.1073/pnas.2113845119
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发表时间：
2022-01-25
影响因子：
11.1
通讯作者：
Hastings MH
中科院分区：
文献类型：
--
作者：
Smyllie NJ;Bagnall J;Koch AA;Niranjan D;Polidarova L;Chesham JE;Chin JW;Partch CL;Loudon ASI;Hastings MH
视交叉上核（SCN）是哺乳动物大脑的主生物钟，协调整个生物体的细胞时钟，以调节生理和行为的日常节奏。SCN计时围绕转录/翻译反馈环旋转，由此PERIOD（PER）和CYPTOCHROME（CRY）蛋白结合并进入细胞核以抑制其自身的表达。PER和CRY的个体和交互行为以及调节它们的机制知之甚少。我们结合了内源性PER 2和病毒载体表达的CRY在SCN切片中的荧光成像，并显示了PER 2如何通过其C末端起作用，控制PER 2的核定位和移动性，以剂量依赖性方式启动SCN计时并控制其周期。我们的研究结果揭示了PER和CRY相互作用的核心SCN钟表。视交叉上核（SCN）的20，000个细胞是哺乳动物大脑的主生物钟，协调整个生物体的下属细胞时钟，驱动生理和行为的适应性日常节奏。SCN计时的典型模型围绕转录/翻译反馈环（TTFL），其中PERIOD（PER）和CRY（CRY）时钟蛋白缔合并易位到细胞核以抑制它们自身的表达。PER和CRY在SCN细胞环境中的基本个体和相互作用行为以及调节它们的机制知之甚少。因此，我们使用共聚焦成像来探索内源性PER 2在PER 2：：Venus报告小鼠的SCN中的行为，所述小鼠用表达各种形式的PER 1和PER 2的病毒载体转导。与野生型SCN中的核定位相反，在不存在CRY蛋白的情况下，PER 2主要在细胞质中并且更具移动的，如通过光漂白后的荧光恢复所测量的。病毒表达的PER 1或PER 2重新定位到细胞核，启动SCN昼夜节律，并确定其周期。我们使用翻译转换来控制PER 1细胞丰度，并发现低水平的PER 1导致PER 2的最小重新定位，但值得注意的是，足以启动和维持昼夜节律。重要的是，C-末端尾是必需的PER 1本地化PER 2的核，并启动SCN的节奏。而在BER 1缺失的SCN中，BER 1 Δ尾对抗PER 2的核定位，相应缩短SCN周期。通过对CRY蛋白的操纵，我们已经深入了解了位于TTFL分子机制核心的PER和CRY的时空行为。
The suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinates cellular clocks across the organism to regulate daily rhythms of physiology and behavior. SCN timekeeping pivots around transcriptional/translational feedback loops whereby PERIOD (PER) and CRYPTOCHROME (CRY) proteins associate and enter the nucleus to inhibit their own expression. The individual and interactive behaviors of PER and CRY and the mechanisms that regulate them are poorly understood. We combined fluorescence imaging of endogenous PER2 and viral vector–expressed CRY in SCN slices and show how CRYs, acting via their C terminus, control nuclear localization and mobility of PER2 to dose-dependently initiate SCN timekeeping and control its period. Our results reveal PER and CRY interactions central to the SCN clockwork. The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.
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