Quantification of protein dynamics driving the circadian clock

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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.