Quantification of protein dynamics driving the circadian clock

生物钟对我们旋转的星球上的生命是必不可少的，在所有生物体中，生物体都驱动着24小时的生理和行为模式，使它们适应光明的规则变化：黑暗周期。在过去的20年里，我们对驱动各种生物体生物钟的遗传机制的理解发生了一场革命，这使得我们对少数核心时钟基因如何调节细胞的起搏有了新的理解。与我们新的基因理解相反，我们几乎不知道这些基因编码的关键蛋白质实际上是如何运作的，以便组装成一个日常的计时器。这种无知是因为我们缺乏必要的技术工具来研究这些蛋白质如何在时间和空间上在细胞内移动。此外，我们一直无法测量生物钟周期不同阶段时钟蛋白的绝对浓度：我们知道这些蛋白每天都会出现和消失，但我们不知道它们在细胞中如何移动，也不知道它们在24小时内的浓度。这是一个非常大的知识鸿沟，因为我们不知道哪些蛋白是限速蛋白，它们如何相互作用，以及重要的是，当基因突变导致核心时钟工作速度发生深刻变化的细胞中，这些蛋白发生了什么。这是需要获得的重要知识，因为在现代生活中，许多人面临着生物钟的重大挑战，导致睡眠异常和新陈代谢副作用。新的计时药学方法，根据人体的时钟系统对药物释放进行计时，是解决这些问题的一种方法，但要做到这一点，我们需要了解时钟机制。事实上，我们实验室的早期工作已经在使用这些方法治疗炎症性疾病方面取得了重大进展。在这个项目中，我们建立在最近的一项研究的基础上，在该研究中，我们使用尖端显微技术实时测量了单细胞中核心时钟蛋白(PER2)在昼夜节律周期中的动态变化。现在，我们建议使用一种新的高效的基因编辑方法来将荧光分子连接到几个关键的时钟基因上。这些分子发射特定波长的光，因此通过使用附着在大约5个关键时钟蛋白上的不同变体，我们可以同时跟踪单个细胞中的几个时钟蛋白。其他方法将使我们能够估计这些蛋白质何时结合形成功能复合体(即相互作用)，并估计它们的浓度。然后，我们将研究它们在大脑中称为视交叉上核(SCN)的关键起搏结构中的行为。SCN协调和同步主要器官的多种身体节律与睡眠/清醒周期，对正常健康至关重要。我们将把这些研究扩展到其他细胞和组织，包括成纤维细胞(所有身体器官中的一种常见细胞类型)。我们将对细胞应用药物和环境刺激，如温度周期，以操纵它们的时钟，并实时监测由此产生的时钟蛋白质的行为。由此，我们将对控制昼夜节律起搏器的中枢机制有重要的新认识。最后，我们的建议将为生物钟计时领域产生无与伦比的资源基础，导致数量生物学的转变，在这种转变中，我们将能够使用数学模型来预测时钟将如何响应环境破坏、疾病等。这是基本的知识，因为它将指导时间药理学领域的未来发展。

Circadian clocks are essential to life on our rotating planet, and in all living organisms drive 24h patterns of physiology and behaviour that adapt them to the regular changes of the light:dark cycle. The past 20 years has witnessed a revolution in our understanding of the genetic mechanisms driving the circadian clock in a wide range of organisms, and this has led to a new understanding of how a small number of core "clock genes" regulate cellular pace-making. In contrast to our new genetic understanding, we know almost nothing of how the key proteins that are encoded by these genes actually behave in order to assemble a daily timekeeper. This ignorance is because we have lacked the essential technological tools to study how these proteins move around the cell in time and space. Also, we have been unable to measure the absolute concentration of clock proteins at different phases of the circadian cycle: we know the proteins appear and disappear on a daily basis, but not how they move around the cell, nor their concentrations, individually and one relative to another, across 24 h. This is a really big gap in knowledge, since we do not know which proteins are rate-limiting, how they interact with each other and importantly, what happens to these proteins in cells in which genetic mutations lead to profound changes in the pace of the core clockwork. This is important knowledge to obtain as in modern life many people are confronted with significant challenges to their circadian clock, leading to abnormal sleep and metabolic side effects. New chrono-pharmaceutical approaches, timing drug delivery to work with the body's clockwork, are one way to address these issues, but to do so we need to understand the clock mechanism. Indeed, earlier work by our laboratory has already made significant advances in the use of these approaches in inflammatory disease. In this project, we build on a recent study in which we used cutting-edge microscopic techniques to measured the dynamic changes in a core clock protein (PER2) in real-time over the circadian cycle in single cells. Now, we propose to use a new efficient method of gene editing to attach fluorescent molecules to several key clock genes. These molecules emit light at a specific wavelength, so by using different variants attached to the 5 or so key clock proteins, we can track several clock proteins simultaneously in individual cells. Other methods will allow us to estimate when these proteins join to form a functional complex (i.e. interact) and also estimate their concentration. We will then study their behaviour in a critical pacemaking structure in the brain called the suprachiasmatic nucleus (SCN). The SCN co-ordinates and synchronises multiple body rhythms in major organs with the sleep/wake cycle, and is crucial for normal health. We will extend these studies to other cells and tissues, including fibroblasts (a common cell type in all body organs). We will apply drugs and environmental stimuli such as temperature cycles to cells to manipulate their clocks, and monitor the resulting behaviour of the clock proteins in real-time. From this, we shall gain important new insight into the central mechanisms controlling the circadian pacemaker. Finally, our proposal will generate for the field of circadian timing an un-paralleled resource base, leading to a transformation in quantitative biology in which we will be able to use mathematical modelling to predict how the clock will behave in response to environmental disruption, disease etc. This is essential knowledge, as it will guide future developments in the field of chronopharmacology.