Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

Abstract

The current model of the mammalian circadian oscillator is predominantly based on data from genetics and biochemistry experiments, while the cell biology of circadian clocks is still in its infancy. Here, we describe a new strategy for the efficient generation of knock-in reporter cell lines using CRISPR technology that is particularly useful for lowly or transiently expressed genes, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins, which allowed to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator.Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.Recent biochemical studies with mouse liver lysates suggest that during the repressive phase, essentially all nuclear PER and CRY proteins are coordinated together in one large repressive complex, with only a minor amount of CRY1 monomers detectable (17). Again, double knock-out of either Per1/2 or Cry1/2 completely prevented the formation of this repressive complex.Most of the current knowledge of PER and CRY protein dynamics resulted either from biochemical data with mixed lysates of many thousand cells, or from single-cell imaging of over-expressed fluorescent tagged fusion proteins (12,13,17,18). Both approaches have clear limitations: Population samplinge.g. cell fractionation followed by Western Blot, chromatography or immunoprecipitation -not only conceals spatial information but also suffers from much reduced temporal resolution. Most importantly, however, population sampling averages signals from thousands of cells thereby masking individual cell properties (e.g. regarding circadian period, phase and amplitude) and degree of noise.While fluorescent tagged proteins constitute an outstanding tool to monitor protein expression and localization in individual living cells, overexpression of PER-and CRY-proteins in most cases disrupts the circadian oscillator and data from such experiments have to be conceived with caution (19,20).Such limitations can be overcome by incorporating a fluorescent tag directly into the proteins' genomic locus. In this case, expression dynamics and level of the resulting fusion protein often remain similar to the wild-type protein and the clock stays intact. Indeed, the PER2-Luciferase and the PER2-Venus knock-in mice -in which PER2 is tagged at the genomic level with ...

Cryptochrome proteins regulate the circadian intracellular behavior and localization of PER2 in mouse suprachiasmatic nucleus neurons

“…Although the mRuby3 tag has a slower folding time than Venus (27), this would not account for such a large phase difference. Recently, a similar phase relationship between PER2 and CRY1 was reported in CRISPR knock-in cell lines (28). This phase lag implies that PER2 and CRY1 will exert serial and not simultaneous actions within the TTFL.…”

Nonlinear phenomena in models of the circadian clock

“…Here, BMAL1 (in complex with other proteins) drives the E-box dependent expression of clock genes, including PER, CRY, REVERB, ROR and DBP. CRY, alone and in complex with PER, inhibits BMAL1 activity (and other E-box containing genes) after a time delay [58,59]. At the same time, the BMAL1 and PER : CRY complexes can be inactivated and degraded [50,51].…”

“…However, changes in the degradation rate of CRY (γ c ) revealed more complex nonlinear phenomena (figure 4, electronic supplementary material, video A1). CRY plays an essential role in the circadian clock: by itself, it represses E-boxes; and together with PER, it contributes to the inhibition of CLOCK : BMAL1 by a number of post-translational processes and nuclear export of the macromolecular complex [53,58,59]. Moreover, the differential role of homologues (CRY1, CRY2) as well as the severe arrhythmic phenotypes of CRY mutants stresses the biological relevance of CRY in the regulation of circadian oscillations [35,58].…”

“…CRY plays an essential role in the circadian clock: by itself, it represses E-boxes; and together with PER, it contributes to the inhibition of CLOCK : BMAL1 by a number of post-translational processes and nuclear export of the macromolecular complex [53,58,59]. Moreover, the differential role of homologues (CRY1, CRY2) as well as the severe arrhythmic phenotypes of CRY mutants stresses the biological relevance of CRY in the regulation of circadian oscillations [35,58]. This motivated us to analyse the effect of changes in γ c on the oscillatory behaviour of the model.…”

Combined multiple transcriptional repression mechanisms generate ultrasensitivity and oscillations

“…blocking type) [15–17]. Because Cry1 displays a delayed expression phase compared to Per, the blocking repression occurs at the late phase, which turns out to be critical for rhythm generation [66–68]. It would be interesting in future work to extend the model to include multiple repressors (e.g.…”