Molecular mechanisms and novel genes mediating temperature compensation in circadian clock neurons of Drosophila melanogaster

果蝇生物钟神经元介导温度补偿的分子机制和新基因

• 批准号: 326244655
• 负责人: Professor Dr. Ralf Stanewsky
• 金额: --
• 依托单位: Institut für Neuro- und Verhaltensbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/326244655?language=en
• 关键词: Molecular; mechanisms; novel; genes; mediating

Circadian clocks are endogenous oscillators, able to control biological rhythms in a constant environment with a period of ca. 24 h. These biological timers are exquisitely sensitive to temperature, because small temperature changes between day and night result in robust clock synchronization. In contrast, their self-sustained 24 h period is independent of its surrounding temperatures, i.e., circadian clocks are temperature compensated. This is a remarkable feature, because all other biological processes speed up with increasing temperatures. It is also essential, because a clock that changes its speed with temperature is no accurate timer. Most organisms are poikilothermic, and therefore heavily depend on proper temperature compensation of their circadian clocks. However, the buffering of the oscillator against temperature changes (temperature compensation) is molecularly not well understood. In the first funding period, we showed that nuclear export of two key Drosophila circadian clock proteins is important for temperature compensation. Our results support the Hastings and Sweeney model for temperature compensation (1957). It assumes that two reactions showing the normal temperature dependent rate increase can nevertheless produce oscillations with constant period length, as long as the second reaction inhibits the first. In Drosophila, this implies that higher rates of nuclear import at warm temperatures would be ‘compensated’ by equally increased rates of nuclear export, keeping the period length constant across temperatures. In the next funding period, we aim to confirm this model using live imaging approaches, which allow to distinguish between nuclear and cytoplasmic localization of clock proteins at different temperatures. We could also show that Casein kinase 1ε (CK1ε, or DBT), which phosphorylates PERIOD and regulates its stability, plays an important role in temperature compensation. Interestingly, we found that a PERIOD mutation interfering with nuclear export shows a phosphorylation defect, specifically at warm temperatures, and a CK1ε mutation that affects temperature compensation in mammals and flies strongly enhances the temperature compensation phenotype of this mutation. We will therefore analyse the function of CK1ε and its interaction with PERIOD for temperature compensation in the next funding period. We could also show that other, so far unknown proteins that function in temperature compensation are subject to nuclear export. Moreover, it is expected that other kinases and proteins are important for temperature compensation. We will therefore perform a genetic screen of ~ 200 isogenic lines generated from natural variants caught in the wild, as well as a candidate screen targeting the known Drosophila kinases. Combined, these approaches will significantly increase our understanding of one of the most fundamental characteristics of circadian clocks.

生物钟是内源性振荡器，能够在大约24小时的恒定环境中控制生物节律。这些生物计时器对温度非常敏感，因为昼夜之间的微小温度变化导致了强大的时钟同步。相反，它们自我维持的24小时周期与周围的温度无关，也就是说，生物钟是温度补偿的。这是一个显著的特征，因为所有其他生物过程都随着温度的升高而加速。这也是必不可少的，因为随着温度变化速度的时钟不是精确的计时器。大多数生物都是变温的，因此在很大程度上依赖于它们生物钟的适当温度补偿。然而，振荡器对温度变化的缓冲（温度补偿）在分子上还没有得到很好的理解。在第一个资助期，我们发现两个关键的果蝇生物钟蛋白的核输出对温度补偿很重要。我们的结果支持Hastings和Sweeney温度补偿模型（1957）。它假定，只要第二个反应抑制第一个反应，两个表现出正常温度依赖性速率增加的反应仍然可以产生具有恒定周期长度的振荡。在果蝇中，这意味着在温暖温度下较高的核输入速率将被同样增加的核输出速率“补偿”，从而保持不同温度下的周期长度不变。在下一个资助期内，我们的目标是使用实时成像方法确认该模型，该方法可以区分时钟蛋白在不同温度下的核和细胞质定位。我们还可以证明酪蛋白激酶1ε （CK1ε，或DBT）磷酸化周期并调节其稳定性，在温度补偿中起重要作用。有趣的是，我们发现干扰核输出的PERIOD突变显示出磷酸化缺陷，特别是在温暖的温度下，而影响哺乳动物和苍蝇温度补偿的CK1ε突变强烈增强了该突变的温度补偿表型。因此，我们将在下一个资助期内分析CK1ε的功能及其与PERIOD的相互作用。我们还可以证明，到目前为止，在温度补偿中起作用的其他未知蛋白质也受到核输出的影响。此外，预计其他激酶和蛋白质在温度补偿中也很重要。因此，我们将对从野外捕获的自然变异体中产生的约200个等基因系进行遗传筛选，以及针对已知果蝇激酶的候选筛选。结合起来，这些方法将大大增加我们对生物钟最基本特征之一的理解。