Quantification of protein dynamics driving the circadian clock

生物钟对我们这个旋转的星球上的生命至关重要，在所有生物体中，生物钟驱动着24小时的生理和行为模式，使它们适应昼夜周期的规律变化。在过去的20年里，我们对多种生物体内驱动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“生物钟基因”如何调节细胞节律有了新的认识。与我们对基因的新理解相反，我们几乎不知道这些基因编码的关键蛋白质是如何运作的，以便组装一个日常计时器。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周围移动。此外，我们还无法测量生物钟蛋白在昼夜周期不同阶段的绝对浓度：我们知道蛋白质每天出现和消失,但不是他们如何移动的细胞,也不是他们的浓度,单独和一个相对于另一个,在24 h。这是一个非常大的差距的知识,因为我们不知道哪些蛋白质病原,它们如何相互作用和重要的是,这些蛋白质在细胞中发生了什么基因突变导致的步伐深刻变化的核心。这是一项重要的知识，因为在现代生活中，许多人的生物钟面临着重大挑战，导致睡眠异常和代谢副作用。新的计时药物方法，即定时给药以配合人体的生物钟，是解决这些问题的一种方法，但要做到这一点，我们需要了解生物钟的机制。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，我们以最近的一项研究为基础，在这项研究中，我们使用尖端的显微技术来实时测量单细胞中核心时钟蛋白（PER2）在昼夜周期中的动态变化。现在，我们建议使用一种新的有效的基因编辑方法，将荧光分子附着在几个关键的时钟基因上。这些分子发出特定波长的光，因此，通过使用附着在5个左右关键时钟蛋白上的不同变体，我们可以在单个细胞中同时跟踪几个时钟蛋白。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物（即相互作用），并估计它们的浓度。然后，我们将研究他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协调和同步睡眠/觉醒周期中主要器官的多种身体节律，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（所有身体器官中常见的细胞类型）。我们将使用药物和环境刺激（如温度周期）来操纵细胞的时钟，并实时监测时钟蛋白的行为。由此，我们将对控制昼夜节律起搏器的中心机制获得重要的新见解。最后，我们的建议将为昼夜节律计时领域产生一个无与伦比的资源基础，导致定量生物学的转变，其中我们将能够使用数学模型来预测时钟如何响应环境破坏，疾病等。这是重要的知识，因为它将指导未来时间药理学领域的发展。

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.