CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping
2021; Springer Nature; Volume: 40; Issue: 7 Linguagem: Inglês
10.15252/embj.2020106745
ISSN1460-2075
AutoresMarrit Putker, David Wong, Estere Seinkmane, Nina M. Rzechorzek, Aiwei Zeng, Nathaniel P. Hoyle, Johanna E. Chesham, Mathew D. Edwards, Kevin A. Feeney, Robin Fischer, Nicolai Peschel, Ko‐Fan Chen, Michael Vanden Oever, Rachel S. Edgar, Christopher P. Selby, Aziz Sancar, John S. O’Neill,
Tópico(s)Photoreceptor and optogenetics research
ResumoArticle25 January 2021Open Access Source DataTransparent process CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping Marrit Putker Marrit Putker orcid.org/0000-0001-9290-408X MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author David C S Wong David C S Wong orcid.org/0000-0002-1712-9527 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Estere Seinkmane Estere Seinkmane orcid.org/0000-0002-3636-4709 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nina M Rzechorzek Nina M Rzechorzek orcid.org/0000-0003-3209-5019 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Aiwei Zeng Aiwei Zeng orcid.org/0000-0003-0354-2529 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nathaniel P Hoyle Nathaniel P Hoyle MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Johanna E Chesham Johanna E Chesham orcid.org/0000-0002-8981-2667 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Mathew D Edwards Mathew D Edwards orcid.org/0000-0002-3573-0025 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kevin A Feeney Kevin A Feeney orcid.org/0000-0003-3143-818X MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Robin Fischer Robin Fischer Biozentrum Universität, Würzburg, Germany Search for more papers by this author Nicolai Peschel Nicolai Peschel orcid.org/0000-0003-3488-832X Biozentrum Universität, Würzburg, Germany Search for more papers by this author Ko-Fan Chen Ko-Fan Chen orcid.org/0000-0002-7305-6254 Institute of Neurology, University College London, London, UK Search for more papers by this author Michael Vanden Oever Michael Vanden Oever orcid.org/0000-0002-2725-6300 Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Rachel S Edgar Rachel S Edgar orcid.org/0000-0002-3348-0851 Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Christopher P Selby Christopher P Selby Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author Aziz Sancar Aziz Sancar orcid.org/0000-0001-6469-4900 Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author John S O'Neill Corresponding Author John S O'Neill [email protected] orcid.org/0000-0003-2204-6096 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Marrit Putker Marrit Putker orcid.org/0000-0001-9290-408X MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author David C S Wong David C S Wong orcid.org/0000-0002-1712-9527 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Estere Seinkmane Estere Seinkmane orcid.org/0000-0002-3636-4709 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nina M Rzechorzek Nina M Rzechorzek orcid.org/0000-0003-3209-5019 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Aiwei Zeng Aiwei Zeng orcid.org/0000-0003-0354-2529 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nathaniel P Hoyle Nathaniel P Hoyle MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Johanna E Chesham Johanna E Chesham orcid.org/0000-0002-8981-2667 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Mathew D Edwards Mathew D Edwards orcid.org/0000-0002-3573-0025 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kevin A Feeney Kevin A Feeney orcid.org/0000-0003-3143-818X MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Robin Fischer Robin Fischer Biozentrum Universität, Würzburg, Germany Search for more papers by this author Nicolai Peschel Nicolai Peschel orcid.org/0000-0003-3488-832X Biozentrum Universität, Würzburg, Germany Search for more papers by this author Ko-Fan Chen Ko-Fan Chen orcid.org/0000-0002-7305-6254 Institute of Neurology, University College London, London, UK Search for more papers by this author Michael Vanden Oever Michael Vanden Oever orcid.org/0000-0002-2725-6300 Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Rachel S Edgar Rachel S Edgar orcid.org/0000-0002-3348-0851 Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Christopher P Selby Christopher P Selby Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author Aziz Sancar Aziz Sancar orcid.org/0000-0001-6469-4900 Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA Search for more papers by this author John S O'Neill Corresponding Author John S O'Neill [email protected] orcid.org/0000-0003-2204-6096 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Marrit Putker1, David C S Wong1, Estere Seinkmane1, Nina M Rzechorzek1, Aiwei Zeng1, Nathaniel P Hoyle1, Johanna E Chesham1, Mathew D Edwards1,6, Kevin A Feeney1, Robin Fischer2, Nicolai Peschel2, Ko-Fan Chen3,7, Michael Vanden Oever4, Rachel S Edgar4, Christopher P Selby5, Aziz Sancar5 and John S O'Neill *,1 1MRC Laboratory of Molecular Biology, Cambridge, UK 2Biozentrum Universität, Würzburg, Germany 3Institute of Neurology, University College London, London, UK 4Faculty of Medicine, Imperial College London, London, UK 5Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA 6Present address: UCL Sainsbury Wellcome Centre for Neural Circuits and Behaviour, London, UK 7Present address: Department of Genetics and Genome Biology, University of Leicester, Leicester, UK *Corresponding author. Tel: +44 7739 729425; E-mail: [email protected] The EMBO Journal (2021)40:e106745https://doi.org/10.15252/embj.2020106745 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational. SYNOPSIS CRYPTOCHROME (CRY) proteins are central regulators of the circadian clock transcription/translation feedback loop. The finding that circadian timekeeping persists, albeit with reduced robustness, in CRY-deficient cells and mice suggests that clock gene activity is determined by evolutionarily-conserved post-translational timing mechanisms. CRY-mediated transcriptional feedback is dispensable for circadian timekeeping in mammalian cells, but functions to make rhythms more robust. CRY knockout mice exhibit behavioural rhythmicity under specific environmental conditions. Circadian variation in the abundance of core clock component PER2 is amplified by, but does not require, rhythmic Per2 transcription. CK1 and GSK3 kinases regulate PER2 stability in the absence of CRY. Introduction The adaptive advantage conferred on organisms by anticipation of the 24-h cycle of day and night has selected for the evolution of circadian clocks that, albeit in different molecular forms, are present throughout all kingdoms of life (Rosbash, 2009; Edgar et al, 2012). Circadian rhythms are robust, in that they are "capable of performing without failure under a wide range of conditions" (Merriam-Webster Dictionary, 2020). The mechanism proposed to generate daily timekeeping in mammalian cells is a delayed transcriptional–translational feedback loop (TTFL) that consists of activating transcription factor complexes containing CLOCK and BMAL1 and repressive complexes, containing the BMAL1:CLOCK targets PERIOD and CRYPTOCHROME (reviewed in Dunlap, 1999; Reppert & Weaver, 2002; Takahashi, 2016). Various coupled, but non-essential, auxiliary transcriptional feedback mechanisms are thought to fine-tune the core TTFL and co-ordinate cell-type-specific temporal organisation of gene expression programs; the best characterised being effected by the E-box mediated rhythmic expression of REV-ERBα/β, encoded by the Nr1d1/2 genes (Preitner et al, 2002; Ueda, 2007; Liu et al, 2008; Takahashi, 2016). These auxiliary loops are not considered sufficient to generate circadian rhythms in the absence of the core TTFL (Preitner et al, 2002; Liu et al, 2008). CRY1 and CRY2 operate semi-redundantly as the essential repressors of CLOCK/BMAL1 activity (Ye et al, 2014; Chiou et al, 2016), required for the nuclear import of PER proteins, and together are considered indispensable for circadian regulation of gene expression in vivo as well as in cells and tissues cultured ex vivo (Kume et al, 1999; Sato et al, 2006; Chiou et al, 2016; Ode et al, 2017). Certainly, mice homozygous null for Cry1 and Cry2 do not express circadian behavioural rest/activity cycles under standard experimental conditions (Thresher et al, 1998; Horst & Muijtjens, 1999; Vitaterna et al, 1999). The hypothalamic suprachiasmatic nucleus (SCN) is a central locus for circadian co-ordination of behaviour and physiology, and research over the last two decades has stressed the strong correlation between SCN timekeeping in vivo and its activity when cultured ex vivo (Welsh et al, 2010; Anand et al, 2013). We were therefore intrigued by the observation that roughly half of organotypic SCN slices prepared from homozygous Cry1−/−,Cry2−/− (CRY knockout; CKO) mouse neonates continue to exhibit ~ 20 h (short period) rhythms, observed using the genetically encoded PER2::LUC clock protein::luciferase fusion reporter (Maywood et al, 2011; Ono et al, 2013b), despite having previously been described as arrhythmic (Liu et al, 2007). Moreover, short period circadian rhythms of locomotor activity have previously been reported for CKO mice raised from birth under constant light (Ono et al, 2013a). As CKO SCN oscillations were only observed in cultured neonatal organotypic slices ex vivo, they were suggested to be a network-level SCN-specific rescue by the activity of neuronal circuits, that desynchronise during post-natal development (Welsh et al, 2010; Ono et al, 2013b). In our view, however, these observations are difficult to reconcile with an essential requirement for CRY in the generation of circadian rhythms. Rather, they are more consistent with CRY making an important contribution to circadian rhythm stability and functional outputs, rather than to the timekeeping mechanism per se, as recently shown for the genes Bmal1 and Clock (Landgraf et al, 2016; Ray et al, 2020), which had both previously been thought indispensable for circadian timekeeping in individual cells (Bunger et al, 2000; DeBruyne et al, 2007). This is further supported by reports that constitutive expression of Cry1 in cells and SCN perturbs but does not abolish circadian oscillations (Fan et al, 2007; Chen et al, 2009; Nangle et al, 2014; Edwards et al, 2016). Recent observations have further questioned the need for transcriptional feedback repression to enable cellular circadian timekeeping. For example, circadian protein translation is regulated by cytosolic BMAL1 through a transcription-independent mechanism (Lipton et al, 2015), and isolated erythrocytes exhibit circadian rhythms despite lacking any DNA (O'Neill & Reddy, 2011; Cho et al, 2014). Moreover, circadian timekeeping in some species of eukaryotic alga and prokaryotic cyanobacteria can occur entirely post-translationally (Sweeney & Haxo, 1961; Nakajima et al, 2005; Tomita et al, 2005; O'Neill et al, 2011). Whether non-transcriptional clock mechanisms operate in other (nucleated) mammalian cells is unknown however, and hence their mechanism and relationship with TTFL-mediated rhythms is an open question. Here, we used cells and tissues from CRY-deficient mice, widely accepted not to exhibit circadian transcriptional regulation (Kume et al, 1999; Ukai-Tadenuma et al, 2011; Edwards et al, 2016) to test whether any timekeeping function remained from which we might begin to dissect the mechanism of the postulated transcription-independent cytosolic oscillator or "cytoscillator" (Hastings et al, 2008). Results Cell-autonomous circadian PER2::LUC rhythms in the absence of CRY proteins Consistent with previous observations, we found no significant circadian organisation of locomotor activity in CRY-deficient (CKO) mice following entrainment to 12 h:12 h light:dark (LD) cycles or in constant light (LL). Upon transition from constant light to constant darkness (DD) [described to be a stronger zeitgeber (Chen et al, 2008)] however, CKO mice expressed rhythmic bouts of consolidated locomotor activity with an average period of ~ 17 h and greater variance than WT controls (Figs 1A and B, and EV1A–C). In Fig 1, representative actograms are plotted as a function of endogenous tau (τ) to allow the periodic organisation of rest–activity cycles to be readily observed; 24-h-plotted actograms are shown in Figs EV1A and EV2A. CKO rhythms under these conditions showed significantly reduced period and amplitude compared with wild-type (WT) controls, but persisted for > 2 weeks, consistent with these mice possessing a residual endogenous biological oscillation that is not entrained by standard environmental light:dark cycles (Fig EV2-EV5 and Appendix). In support of this interpretation, and in accordance with previous reports (Maywood et al, 2011; Ono et al, 2013b), longitudinal bioluminescence recordings of organotypic PER2::LUC SCN slices cultured ex vivo from WT or CKO neonates revealed rhythmic PER2 expression in approximately 40% of CKO slices (Fig 1C). In line with behavioural data and previous reports, these CKO SCN rhythms exhibited significantly shorter periods compared with WT controls (Fig 1D). Figure 1. CRY-independent circadian timekeeping occurs cell-autonomously Representative double-plotted actograms showing wheel-running activity of wild-type (WT) and CRY-deficient (CRY knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness. Note the 48 h x-axis for WT vs. 32 h for CKO. Full figure showing CKO data in modulo 24 h is presented in Fig EV1A. Mean period and amplitude (± SEM) of mouse behavioural data (n = 4). P-values were calculated by two-way ANOVA. Longitudinal bioluminescence recordings of organotypic SCN slices from WT (black) and CKO (red) PER2::LUC mice (RLU; relative light units). Mean period and amplitude (± SEM) of rhythmic SCN bioluminescence traces. P-values were calculated by two-way ANOVA. Circadian PER2::LUC expression in immortalised WT and CKO adult lung fibroblasts. Left panel shows two raw traces of a representative longitudinal bioluminescence recording, and right panel shows same data detrended with a 24-h moving average to remove differences in baseline expression. Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments (n ≥ 3 per experiment, individual values ± SEM shown). P-values were calculated by an unpaired t-test with Welch correction. Standard deviations differ significantly between WT and CKO (F-test: P < 0.0001). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CRY-independent circadian timekeeping occurs cell-autonomously Representative double-plotted actograms showing wheel-running activity of WT and CKO mice during 12 h:12 h light:dark (LD) cycles (yellow shading indicating lights on) (top) or during constant light (LL) (bottom) and thereafter in constant darkness (DD). Top four figures have same x-axis (modulo 24 h). Rhythmic behaviour of CKO mice in LL > DD condition becomes clear when plotting the data in 16-h modulo (i.e. x-axis being 32 h). LD figure WT modulo 24-h and CKO modulo 16-h are also presented in Fig 1A. Bottom left, representative periodograms of WT and CKO mice over 2 weeks in constant darkness, following either 12 h:12 h light:dark cycles or constant light. A second experimental cohort highlights significant differences in period variance (left, horizontal line represents mean, F-test for variance); period and amplitude (right) of CKO (n = 10) vs. WT (n = 12) mice over 2 weeks in constant darkness following 1 week under constant light (mean ± SEM, 2-way ANOVA with Sidak's MCT). Examples of independent bioluminescence recordings of PER2::LUC expression in CRY-deficient fibroblasts showing variability in shape and baseline of rhythmic CKO traces. Two representative traces are shown per experiment. Stringent entrainment, e.g. with temperature cycles or dexamethasone, increases the likelihood of observing rhythmicity, but only in approximately 30% of the experiments did we observe clearly rhythmic expression of PER2::LUC over 3 cycles. Despite our best efforts, over many years, we were unable to identify a set of entrainment and recording conditions that consistently produced CKO PER2::LUC rhythms and we were forced to conclude that more variables were at play than we were adequately able to control for. Genotyping CKO fibroblasts used throughout this study. Left: PCR genotyping shows the expected pattern of CRY1 and CRY2 knockout. Right: Western blot analysis of whole cell lysates (WCL) and probed with antibodies against CRY1 and CRY2. Interexperimental comparison of PER2::LUC periods in WT vs. CKO fibroblasts. Paired comparison of period means of experiments used for Fig 1F where CKO traces were rhythmic. P-values were calculated by paired t-test. Intraexperimental standard deviations (i.e. between replicates) were calculated for all experiments with > 3 rhythmic traces (mean ± SEM). P-value was calculated by unpaired t-test. Damping rates of all individual detrended traces of example experiments shown in Fig EV1E were calculated by damped sine wave fitting (mean ± SEM, WT n = 106, CKO n = 109). P-value was calculated by unpaired t-test with Welch correction. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Entrainment deficiency of CRY-deficient mice to environmental light:dark cycles and Timeless-independent protein rhythms in Drosophila melanogaster A. Representative actograms showing that CKO mice (n = 5) cannot be entrained to 8 h:8 h or 12 h:12 h light:dark cycles, whereas WT mice entrain to 12 h:12 h but not 8 h:8 h light-dark cycles (n = 5). Equal numbers of age-matched male and female mice were used. B, C. For CKO mice during under LD cycles, the dominant period of behavioural rhythms is determined by the period of the zeitgeber, whereas amplitude does not vary significantly between 16-h and 24-h cycles. In contrast, the period of WT behavioural rhythms does not vary significantly between 16-h and 24-h cycles, whereas amplitude is significantly reduced under unnatural 8 h:8 h light:dark cycles. This indicates the robustness conferred by CRY to circadian rhythms of locomotor activity in WT mice in vivo. Two-way ANOVA P-values and significance from Sidak's multiple comparisons test are reported; horizontal line represents mean. D. Normalised, detrended bioluminescence recording of the XLG-luciferase reporter (XLG-LUC; equivalent of PER2::LUC) expressed in WT and timeless knockout (Timout) flies under constant darkness (detrended means ± SEM; WT n = 21, Timout n = 36). Note the difference in y-axis scaling. E. Damped sine wave fit to the data presented in (D). P-values (extra sum-of-squares F-test) indicate comparison of fit test with the null hypothesis (straight line). F. Significant differences in the period and amplitude of XLG-LUC rhythms of Timout compared with WT flies. Mean ± SEM, P-values indicate unpaired t-test, WT n = 21, Timout n = 36. Data information: The generation of Timout flies is reported in Lamaze et al (2017). Similar to CRY-deficient mice, whole gene timeless knockout flies are characterised as being behaviourally arrhythmic under constant darkness following entrainment by light:dark cycles: https://opus.bibliothek.uni-wuerzburg.de/frontdoor/index/index/year/2015/docId/11914 Download figure Download PowerPoint Click here to expand this figure. Figure EV3. CRY-independent rhythms are regulated post-transcriptionally Standard curve of recombinant luciferase that was used to determine the number of PER2::LUC molecules. Known concentrations of recombinant luciferase were spiked into (non-luciferase containing) cell lysate to reproduce experimental conditions, and the luciferase signal was measured with a 20-s integration time (CP 20 s; counts per 20 s). Data were fitted with a straight line (red line, ± 95% CI). The grey dotted lines indicate the (linear) area of the curve used to determine the number of PER2::LUC molecules of the experiment shown in Fig 3A. Western blot analysis of BMAL1 co-immunoprecipitation samples shown in Fig 3B. BMAL1 or control (IgG) pulldowns were performed at the peak of PER2::LUC expression (determined in parallel PER2::LUC recordings) in 3 technical replicates (A–C). Full blots are shown in Source data Fig EV3. Bmal1 mRNA levels were determined by qPCR over one circadian period (n = 2, mean ± SEM). The WT timeseries were preferentially fit with a circadian damped sine wave compared with a straight line (extra sum-of-squares F-test, P = 0.0321), but not the CKO timeseries (ns). PER2::LUC co-recording from parallel cultures is depicted in Fig 3C. Detrended bioluminescence data of transcriptional reporter Cry1:LUC in WT and CKO mouse adult fibroblasts (MAFs) (n = 4, mean ± SEM). WT traces fit circadian damped sine wave over straight line in extra sum-of-squares F-test (P < 0.0001), whereas no sine wave could be fit to CKO traces (no P-value). Detrended Per2 and Nr1d1 promoter activity in WT, CKO and quadruple Cry1/2-Per1/2 knockout (CPKO) mouse embryonic fibroblasts (MEFs) recorded at 32°C, n = 3, mean (solid) ±SEM (dashed). WT Per2 and all Nr1d1 traces were preferentially fit with a circadian damped sine wave over straight line (extra sum-of-squares F-test, P < 0.0001), whereas no sine wave could be fit to the other traces (not significant, no P-value). Period analysis shows that Nr1d1 promoter oscillations are temperature-compensated (mean ± SEM, n = 3). Expanded view of Per2:LUC recordings to show no circadian oscillations of Per2 promoter activity is detected in C(P)KO MEFs, n = 3, mean (solid) ±SEM (dashed). Zoomed-out version of 37°C recording is also shown in Fig 3D and of 32°C recording in Fig EV3E. WT Per2 traces were preferentially fit with a circadian damped sine wave over straight line (extra sum-of-squares F-test, P < 0.0001), whereas no sine wave could be fit to the other traces (not significant, no P-value). Period analysis shows that also WT Per2 promoter oscillations are temperature-compensated, as expected (n = 3, mean ± SEM). 24-h average (from 24 to 48 h) raw luciferase counts from Per2:LUC and Nr1d1:LUC counts demonstrate that reporter expression levels do not explain a difference in amplitude in circadian oscillations (n = 3, mean ± SEM). Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. PER2::LUC stability oscillates in CRY-deficient cells WT and CKO cells were assayed for puromycin incorporation over two circadian cycles. Cells were synchronised by temperature cycles and dexamethasone, and harvested every 3 h after a 10-min puromycin pulse (10 µg/ml). Incorporation was measured by Western blotting with an anti-puromycin antibody. Western blots were quantified and corrected for total protein loading (Coomassie staining). Mean ± SEM (n = 3). Bioluminescence co-recording of puromycin labelling time course shows circadian PER2::LUC expression in both genotypes. Mean ± SEM (n = 3). Western blot analysis of BMAL1 immunoprecipitation with antibodies specific for S6K, eIF4a and BMAL1. Cells were harvested 12 or 24 h after dexamethasone synchronisation, and BMAL1 was immunoprecipitated. Example of a bioluminescence recording of WT PER2::LUC (left) or SV40::LUC (right) cells pulsed with 10 µM CHX after 46 h of recording. The resulting raw data (symbols) were fitted with a one-phase decay curve (blue line). Multiple stable SV40::LUC fibroblast lines with different basal expression levels were treated with 25 μg/ml CHX allowing the turnover of luciferase to be inferred from the decay in bioluminescence signal. Left panel, the decline in luciferase activity was fit with a simple one-phase exponential decay curve (solid lines) to derive the half-life of luciferase in each cell line. Right panel, no significant relationship between the level of luciferase expression and luciferase stability was observed (straight line vs. horizontal line fit P-value is reported, extra sum-of-squares F-test). Timing of CHX pulses (labelled I-II a (cycle 1) and b (cycle 2)), plotted on bioluminescence trace of WT PER2::LUC control cells, corresponding to data presented in G and H. PER2::LUC half-life at different phases in the circadian cycle in WT cells (mean ± SEM). P-values were calculated by two-tailed t-test. Half-life was quantified by one-phase decay line-fitting of bioluminescence traces from CHX pulsed cells. SV40::LUC half-life at different phases in the circadian cycle in fibroblasts (mean ± SEM). P-values were calculated by two-tailed t-test. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. A role for CK1 and GSK3 in the cytoplasmic oscillator Bioluminescence recordings of WT and CKO PER2::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.3 µM; PF), as quantified in Fig 5A (n = 3, detrended mean ± SEM). As in (A), GSK3 inhibitor CHIR99021 (5 µM; CHIR). As in (A), in the presence of CRY turnover inhibitor KL001 (1 µM). Bioluminescence recordings and respective period quantifications of WT and CPKO Nr1d1::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.1 µM) or GSK3 inhibitor CHIR99021 (3 µM; CHIR) (n = 4, detrended mean ± SEM). P-value of the two-way ANOVA (genotype vs. drug interaction effect) is reported. Download figure Download PowerPoint Two explanations might account for the variable CKO SCN phenotype: (i) the previously proposed explanation: genetic loss of function is compensated at a network level by SCN-specific neuronal circuits whose function is sensitive to developmental phase and small variations in slice preparation (Liu et al, 2007; Evans et al, 2012; Ono et al, 2013b; Tokuda et al, 2015); or (ii) CKO (SCN) cells have cell-intrinsic circadian rhythms that are expressed (or observed) more stochastically and with less robustness than their WT counterparts, and can be amplified by SCN interneuronal signalling (Welsh et al, 2010; O'Neill & Reddy, 2012). To distinguish between these two possibilities, we asked whether PER2::LUC rhythms are observed in populations of immortalised PER2::LUC CKO adult fibroblasts, which lack the specialised interneuronal neuropeptidergic signalling that is so essential to SCN amplitude and robustness in and ex vivo (Welsh et al, 2010; O'Neill & Reddy, 2012). We observed this to be the case (Figs 1E, and EV1C and D). Across > 100 recordings, using independently generated cell lines cultured from multiple CRY-deficient mice (male and female), we observed PER2::LUC rhythms that persisted for several days under constant conditions. Again, the mean period of rhythms in CRY-deficient cells was significantly shorter than WT controls, and with increased variance within and between experiments (F-test P-value < 0.0001, Figs 1F, and EV1E and F). Consistent with SCN results, rhythmic PER2::LUC expression in CKO cells occurred stochastically between experiments, being observed in ~ 30% of independently performed assays. Importantly, there was very little variation in the occurrence of rhythmicity w
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