Quantification of protein dynamics driving the circadian clock

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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.