Quantification of protein dynamics driving the circadian clock

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.

Circadian biology has been driven by a revolution in genetics. In marked contrast, we have generally been unable to study the encoded proteins that actually make up the core circadian clockwork in cells: we do not understand how they behave in circadian time, or across cellular space. We need, therefore, new tools to allow accurate quantitation of the properties and behaviour of endogenous clock proteins. This will offer mechanistic understanding and support generation of new models with predictive power in this complex dynamic system, likely serving as an exemplar for quantitative cell biology well beyond the circadian domain.

We aim to build on recent success in which we tagged the murine PER2 gene in-frame with a VENUS fluorophore, and using cells from these mice obtained the first dynamic images and quantitative measures of how an endogenous clock protein behaves. This has already led to a re-appraisal of current models for circadian pacemaking.

To further develop this breakthrough we shall use CRISPR gene editing to create lines of mice in which circadian proteins (CRY1, CRY2, BMAL1, PER2) are marked with spectrally distinct fluorophores (e.g. Venus, Cerulean, mKate). We shall use Fluorescence Correlation Spectroscopy to quantify protein concentrations over the cycle and Fluorescence Cross-Correlation Spectroscopy to examine protein interactions. Exploiting new insights from structural biology, we shall test the roles of domains implicated in complex formation and circadian function. We will study the effects of mutations that alter the circadian pacemaker and explore how pharmacological manipulation drives the behaviour of clock complexes. We shall examine clock proteins in fibroblasts and SCN clock neurons to define common and divergent mechanisms.

The research questions posed within this proposal are of major interest to ACADEMIC GROUPINGS in Biological, Biomedical Sciences, Clinical science, Biomathematics and Light-microscopy Development. The academic community will benefit from elucidation of the cellular and molecular mechanisms whereby circadian clock proteins interact to generate a daily cycles of gene expression and cellular function. As such, research findings will impact greatly in the field of quantitative cell biology. In addition, longer-term understanding of the circadian clock mechanism will inform chronotherapeutic approaches to the management and treatment of disease and ultimately inform the HEALTH CARE COMMUNITY of the value of environmental regularity in patient management. We will disseminate findings by publishing primary papers and reviews in high impact journals, and presenting work at national and international meetings. We anticipate that the proposed work will produce 2-4 high-quality primary research papers.

Our findings will be of high interest to the GENERAL PUBLIC, which is acutely aware of the day-to-day importance of a regular schedule for mental and physical well being, and is very engaged when hearing about the latest progress in understanding the neural, cellular and molecular genetic bases to the "body clock". At its most basic, the work will engage sections of the population who wish to learn about their health and human physiology in the context regular sleep, eating patterns, exercise routines etc: how is this controlled on a cellular level? Research findings will be delivered to the general public through public engagement activities (e.g. annual science open days at the UoM and Cambridge, Café Scientifique, Soapbox Science presentations), as well as through mass media. Both ASIL and MHH have conducted numerous interviews: for example, several of our recent papers have been reported widely in national and international newspapers, on local radio, and on the intranet following press releases issued by the University of Manchester, the LMB and the BBSRC and MRC.

The proposed research is of interest to PHARMACEUTICAL COMPANIES due to direct implications of the circadian clock for human disease. In the context of "building partnerships to enhance take-up and impact, thereby contributing to the economic competitiveness of the United Kingdom", our laboratories have taken a major lead within the extensive community of researchers at the University of Manchester by developing significant interactions and links with GSK and a joint nuclear receptor Biology Programme. In parallel the LMB has recently embarked on a programme of "blue-skies" collaborative work with Astra-Zeneca/ MedImmune, the world head-quarters and new central research facility of which are now being constructed adjacent to the new LMB building on Cambridge Biomedical Campus.