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The ∼20,000 cells of the suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinate subordinate cellular clocks across the organism, driving adaptive daily rhythms of physiology and behavior. The canonical model for SCN timekeeping pivots around transcriptional/translational feedback loops (TTFL) whereby PERIOD (PER) and CRYPTOCHROME (CRY) clock proteins associate and translocate to the nucleus to inhibit their own expression. The fundamental individual and interactive behaviors of PER and CRY in the SCN cellular environment and the mechanisms that regulate them are poorly understood. We therefore used confocal imaging to explore the behavior of endogenous PER2 in the SCN of PER2::Venus reporter mice, transduced with viral vectors expressing various forms of CRY1 and CRY2. In contrast to nuclear localization in wild-type SCN, in the absence of CRY proteins, PER2 was predominantly cytoplasmic and more mobile, as measured by fluorescence recovery after photobleaching. Virally expressed CRY1 or CRY2 relocalized PER2 to the nucleus, initiated SCN circadian rhythms, and determined their period. We used translational switching to control CRY1 cellular abundance and found that low levels of CRY1 resulted in minimal relocalization of PER2, but yet, remarkably, were sufficient to initiate and maintain circadian rhythmicity. Importantly, the C-terminal tail was necessary for CRY1 to localize PER2 to the nucleus and to initiate SCN rhythms. In CRY1-null SCN, CRY1Δtail opposed PER2 nuclear localization and correspondingly shortened SCN period. Through manipulation of CRY proteins, we have obtained insights into the spatiotemporal behaviors of PER and CRY sitting at the heart of the TTFL molecular mechanism.

The mammalian circadian clock exerts control of daily gene expression through cycles of DNA binding. Here, we develop a quantitative model of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We used quantitative imaging to track dynamic changes in endogenous labelled proteins across peripheral tissues and the SCN. We determine the contribution of multiple rhythmic processes coordinating BMAL1 DNA binding, including cycling molecular abundance, binding affinities, and repression. We find nuclear BMAL1 concentration determines corresponding CLOCK through heterodimerisation and define a DNA residence time of this complex. Repression of CLOCK:BMAL1 is achieved through rhythmic changes to BMAL1:CRY1 association and high-affinity interactions between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

The biological clock of the suprachiasmatic nucleus (SCN) orchestrates circadian (approximately daily) rhythms of behaviour and physiology that underpin health. SCN cell‐autonomous time‐keeping revolves around a transcriptional/translational feedback loop (TTFL) within which PERIOD (PER1,2) and CRYPTOCHROME (CRY1,2) proteins heterodimerise and suppress trans‐activation of their encoding genes (Per1,2; Cry1,2). To explore its contribution to SCN time‐keeping, we used adeno‐associated virus–mediated translational switching to express PER2 (tsPER2) in organotypic SCN slices carrying bioluminescent TTFL circadian reporters. Translational switching requires provision of the non‐canonical amino acid, alkyne lysine (AlkK), for protein expression. Correspondingly, AlkK, but not vehicle, induced constitutive expression of tsPER2 in SCN neurons and reversibly and dose‐dependently suppressed pPer1‐driven transcription in PER‐deficient (Per1,2‐null) SCN, illustrating the potency of PER2 in negative regulation within the TTFL. Constitutive expression of tsPER2, however, failed to initiate circadian oscillations in arrhythmic PER‐deficient SCN. In rhythmic, PER‐competent SCN, AlkK dose‐dependently reduced the amplitude of PER2‐reported oscillations as inhibition by tsPER2 progressively damped the TTFL. tsPER2 also dose‐dependently lengthened the period of the SCN TTFL and neuronal calcium rhythms. Following wash‐out of AlkK to remove tsPER2, the SCN regained TTFL amplitude and period. Furthermore, SCN retained their pre‐washout phase: the removal of tsPER2 did not phase‐shift the TTFL. Given that constitutive tsCRY1 can regulate TTFL amplitude and period, but also reset TTFL phase and initiate rhythms in CRY‐deficient SCN, these results reveal overlapping and distinct properties of PER2 and CRY1 within the SCN, and emphasise the utility of translational switching to explore the functions of circadian proteins. [ABSTRACT FROM AUTHOR]

It has been 50 years since the suprachiasmatic nucleus (SCN) was first identified as the central circadian clock and 25 years since the last overview of developments in the field was published in the Journal of Biological Rhythms. Here, we explore new mechanisms and concepts that have emerged in the subsequent 25 years. Since 1997, methodological developments, such as luminescent and fluorescent reporter techniques, have revealed intricate relationships between cellular and network-level mechanisms. In particular, specific neuropeptides such as arginine vasopressin, vasoactive intestinal peptide, and gastrin-releasing peptide have been identified as key players in the synchronization of cellular circadian rhythms within the SCN. The discovery of multiple oscillators governing behavioral and physiological rhythms has significantly advanced our understanding of the circadian clock. The interaction between neurons and glial cells has been found to play a crucial role in regulating these circadian rhythms within the SCN. Furthermore, the properties of the SCN network vary across ontogenetic stages. The application of cell type–specific genetic manipulations has revealed components of the functional input-output system of the SCN and their correlation with physiological functions. This review concludes with the high-risk effort of identifying open questions and challenges that lie ahead. [ABSTRACT FROM AUTHOR]

Remembering when it was last able to eat helps an animal optimise its foraging strategy for future meals. But where is that time memory located? A new study now shows that it is embedded in an enigmatic, light-entrainable circadian (daily) clock. [ABSTRACT FROM AUTHOR]