PER2 and CRY1, key body clock proteins in the brain’s suprachiasmatic nucleus, act more independently than previously thought – challenging the long-held model of circadian timekeeping
The suprachiasmatic nucleus (SCN) is a network of cells in the brain responsible for governing the body’s circadian rhythms, acting as a pacemaker to control daily cycles of behaviour and physiology. SCN timekeeping is powered by a group of proteins operating in a repeating loop called the transcriptional-translational feedback loop (TTFL). To better understand how this system works, Michael Hastings’s group, in the LMB’s Neurobiology Division, have analysed the behaviours of the TTFL proteins Period (PER), Cryptochrome (CRY) and BMAL1. The study, led by Investigator Scientist Nicola Smyllie, found that these proteins act far more independently than previously thought, revealing a previously unappreciated complexity to the TTFL model.
To directly image the proteins in question, the team developed reporter mice in which PER2, CRY1 and BMAL1 were tagged with fluorescent markers, enabling real-time monitoring of these proteins in live SCN tissue slices. Using high-resolution confocal microscopy, the team were able to examine the localisation, mobility and interactions of the proteins across several circadian cycles with cellular rhythms maintained across eight days. The team also treated the SCN slices with compounds to stabilise PER and CRY1 to monitor the impacts on TTFL function and overall circadian timekeeping.
This revealed a surprising level of disparity in the spatial, behavioural and molecular properties of each protein. PER2 was far more mobile and was more commonly localised to the cytoplasm of the cell, while CRY1 and BMAL1 were predominantly located in the nucleus (with BMAL1 likely bound to DNA) and more stable. Interestingly, CRY1 and BMAL1 peaked in abundance six to seven hours after PER2, contradicting the previous assumption that the repressor proteins CRY1 and PER2 appeared and acted together.
The proteins were also found to have significantly differing half-lives; PER2 exhibited the shortest at roughly two hours, whilst BMAL1 had the longest and remained for several days. Pharmacological intervention to stabilise PER2 and CRY1 was found to lengthen the circadian period, but in distinct ways; PER2 affected the phase relationship between clock proteins, whereas stabilised CRY1 more dramatically suppressed TTFL-driven transcription, again usurping the pre-existing notion of PER2 and CRY1 acting in unison.
This work overhauls our understanding of the TTFL model. Rather than acting in a prescribed duet, the divergence in behaviour and regulation of PER2 and CRY1 instead paint the TTFL more like a complex orchestra, with each protein assuming their own roles with unique rhythms and strengths. Maintaining the given stability and abundance of each protein is crucial in determining the rhythm of the SCN clock.
This study also raises clinical implications, as this new understanding of TTFL could offer potential for treating circadian disruption in health conditions such as sleep disorders and depression. Furthermore, this increased understanding of the molecular machinery of the SCN could help to inform development of targeted chronotherapeutic strategies to help treat problems incurred from jet lag or shift work and during old age.
This work was funded by UKRI MRC and UKRI BBSRC.
Further references
Quantitative measures of clock protein dynamics in the mouse suprachiasmatic nucleus extends the circadian time-keeping model. Smyllie, N.J., Koch, A.A., Adamson, A.D., Patton, A.P., Johnson, A., Bagnall, J.S., Johnson, O., Meng, Q-J., Loudon, A.S.I. and Hastings, M.H. EMBO
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Cryptochrome proteins are integral to maintain time within the brain’s master clock
Advanced understanding of how suprachiasmatic nucleus regulates the body’s circadian rhythm
Animal research statement
As a publicly funded research institute, the LMB is committed to engagement and transparency in all aspects of its research. This research used mice, in accordance with the UK Animals (Scientific Procedures) Act 1986. This work was conducted under a Project Licence, reviewed and approved by the MRC Laboratory of Molecular Biology (LMB) Animal Welfare and Ethical Review Body (AWERB) committee and the UK Home Office.
The LMB uses the minimum number of rodents necessary to achieve results and only uses animals in research where there are no suitable alternatives, in line with the 3R’s (replace, reduce, refine). We currently work with fruit flies, nematode worms, mice, rats and zebrafish.