LAUSR

Significance The suprachiasmatic nucleus (SCN), the master circadian clock of the mammalian brain, coordinates cellular clocks across the organism to regulate daily rhythms of physiology and behavior. SCN timekeeping pivots around transcriptional/translational feedback loops whereby PERIOD (PER) and CRYPTOCHROME (CRY) proteins associate and enter the nucleus to inhibit their own expression. The individual and interactive behaviors of PER and CRY and the mechanisms that regulate them are poorly understood. We combined fluorescence imaging of endogenous PER2 and viral vector–expressed CRY in SCN slices and show how CRYs, acting via their C terminus, control nuclear localization and mobility of PER2 to dose-dependently initiate SCN timekeeping and control its period. Our results reveal PER and CRY interactions central to the SCN clockwork. 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.