Dissection of a novel 'periphery to brain' circuit that synchronizes Drosophila's circadian clock with temperature cycles

生物钟是包括人类在内的大多数生物体内滴答作响的生物计时器。这些生物钟控制着一系列广泛的生物过程，包括我们的睡眠/觉醒周期、食欲或体温。它们在没有任何外界输入的情况下工作，这意味着它们是真正的时钟，以大约24小时的节奏滴答作响，即使生物体处于完全黑暗的环境中。换句话说，即使在与外界完全隔离的情况下，人类也保持着每天的睡眠和觉醒周期。另一方面，在自然界中，我们的生物钟受到环境的强烈影响，例如每天的光/暗和温度变化。因此，昼夜节律与环境同步。关于生物钟的独立性及其与环境沟通的能力，一个令人印象深刻的例子是与跨时区旅行（时差）或轮班工作相关的现象。如果你有“时差”，你的生物钟仍然根据你登机的时间滴答作响，并告诉你在半夜要清醒。渐渐地，你的生物钟会调整（同步）到新的时区，你会再次感到舒适。从分子上讲，生物钟是由几个所谓的“时钟基因”组成的，这些基因活跃于大脑的某些神经元中，控制着重要的生物节律。这些生物钟基因本身的活动是有节奏的——它们每24小时活跃一次。基因活动最大或最小的时间是由生物体所暴露的自然光暗和温度循环决定的。换句话说，我们的生物钟与外界同步的方式是通过直接改变生物钟基因表达来调节的，以响应光/暗或温度的变化。目前的提议旨在研究温度周期如何使果蝇的生物钟同步。我们对解决这个问题非常感兴趣，因为我们发现所涉及的机制必须与光同步所描述的非常不同。在果蝇中，后者主要由蓝光感光体隐花色素（Cry）介导，这是一种在果蝇大脑生物钟神经元中表达的蛋白质。因此，即使将苍蝇的大脑取出并放在培养皿中培养，苍蝇大脑的生物钟也可以通过明暗循环来同步。虽然（与Cry类似）我们最初预计时钟神经元也包含一个温度受体，但我们发现“盘子里的大脑”不能使它们的时钟与温度周期同步！这是一个很大的惊喜，表明大脑中的时钟神经元从苍蝇的其他地方接收温度信息。在这个提议中，我们想要识别这些细胞或器官，我们已经有了一些有希望的初步发现：之前我们已经分离出‘nocte’作为温度同步突变体。当我们减少周围感觉器官中“nocte”基因的功能时，我们可以破坏苍蝇同步温度周期的能力。这意味着我们现在有了一个基因和候选的感觉结构，这将允许我们开始解开温度同步途径。我们还将研究一类离子通道（色氨酸通道），它已被证明在脊椎动物和昆虫中起“环境传感器”的作用。例如，它们可以调节对极端温度、触摸或辣椒的反应。我们将通过分析可用的Trp通道突变体来测试这些通道是否对温度同步很重要。最后，我们希望通过应用涉及质谱分析的现代蛋白质组学纯化方法来鉴定与Nocte相互作用的蛋白质。通过这样做，我们希望确定有助于解决果蝇温度同步途径的其他因素。

Circadian clocks are biological timers that tick in most organisms, including humans. These clocks control a wide array of biological processes, including our sleep/wake cycle, appetite, or body temperature. They function without any input from the outside, meaning they are true clocks that tick with an approximate 24 hr rhythm, even when the organism is kept in total darkness. In other words, human beings keep their daily sleep wake cycles, even when kept in total isolation from the outside world. In nature on the other hand, our circadian clocks are strongly influenced by the environment, as for example by the daily light/dark and temperature changes. As a consequence, circadian rhythms are synchronized with the environment. An impressive example for both the independence of circadian clocks and their ability to communicate with the environment are the phenomena associated with travel across time zones (jetlag) or shift work. If you are 'jetlagged', your circadian clock is still ticking according to the time where you boarded your plane and is telling you to be awake in the middle of the night. Gradually though, your internal clock will adjust (synchronize) to the new time zone and you will feel comfortable again. Molecularly, circadian clocks are assembled by several so called 'clock genes', which are active in certain neurons in the brain and control important biological rhythms. The activity of these clock genes itself is regulated in a rhythmic fashion-they become active in 24 hr periods. The time of maximal or minimal gene activity is determined by the natural light-dark and temperature cycles an organism is exposed to. In other words, the way our clocks are synchronized with the outside world is mediated by directly changing clock gene expression in response to light/dark or temperature changes. The current proposal is aimed to investigate how temperature cycles can synchronize the circadian clock of fruit flies. We are very interested to solve this question, because we discovered that the mechanism involved must be very different from that described for light-synchronization. In flies, the latter is mainly mediated by the blue-light photoreceptor Cryptochrome (Cry), a protein that is expressed within the clock neurons in the fly's brain. As a consequence, the circadian clock of fly brains can be synchronized by light:dark cycles, even when the brains are taken out of the fly and cultured in a dish. Although (in analogy with Cry) we initially expected that the clock neurons also contain a temperature receptor, we found that 'brains in a dish' can not synchronize their clock to temperature cycles! This was a big surprise, indicating that the clock neurons in the brain receive the temperature information from somewhere else in the fly. In this proposal we want to identify these cells or organs, and we already have some promising preliminary findings: Previously we had isolated 'nocte' as temperature synchronization mutant. When we reduce the function of the 'nocte' gene in peripheral sensory organs, we can destroy the fly's ability to synchronize to temperature cycles. This means we now have a gene and candidate sensory structures at hand, which will allow us to start unravelling the temperature synchronization pathway. We will also investigate a class of ion channels (Trp channels), that has been shown to function as 'environmental sensors' in both vertebrates and insects. They can, for example, mediate responses to extreme temperatures, touch, or hot chilli peppers. We will test if these channels are important for temperature synchronization by analysis of available Trp channel mutants. Finally, we want to identify proteins that interact with Nocte by applying a modern proteomics purification approach involving Mass-spec analysis. By doing this, we hope to identify additional factors that will help to resolve the temperature synchronization pathway in flies.