Circadian clocks in human red blood cells - PubMed (original) (raw)

Circadian clocks in human red blood cells

John S O'Neill et al. Nature. 2011.

Abstract

Circadian (∼24 hour) clocks are fundamentally important for coordinated physiology in organisms as diverse as cyanobacteria and humans. All current models of the molecular circadian clockwork in eukaryotic cells are based on transcription-translation feedback loops. Non-transcriptional mechanisms in the clockwork have been difficult to study in mammalian systems. We circumvented these problems by developing novel assays using human red blood cells, which have no nucleus (or DNA) and therefore cannot perform transcription. Our results show that transcription is not required for circadian oscillations in humans, and that non-transcriptional events seem to be sufficient to sustain cellular circadian rhythms. Using red blood cells, we found that peroxiredoxins, highly conserved antioxidant proteins, undergo ∼24-hour redox cycles, which persist for many days under constant conditions (that is, in the absence of external cues). Moreover, these rhythms are entrainable (that is, tunable by environmental stimuli) and temperature-compensated, both key features of circadian rhythms. We anticipate that our findings will facilitate more sophisticated cellular clock models, highlighting the interdependency of transcriptional and non-transcriptional oscillations in potentially all eukaryotic cells.

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Figures

Figure 1

Figure 1. Circadian oscillation of peroxiredoxin (PRX) oxidation in human red blood cells

a, Red blood cells from three human subjects (A, B, C) were entrained by temperature cycles and then kept under constant conditions (at 37°C, in total darkness) and sampled every 4 hours. b, Red blood cells incubated in alternating 12 hour cycles of high (37°C) and low (32°C) temperature. Representative immunoblots showing oxidised/hyperoxidised peroxiredoxin (PRX-SO2/3) dimer are shown with loading controls. Quantification by densitometry is shown below. Values were normalised to the maximum for each blot. Solid line represents mean normalised intensity, with grey lines indicating s.e.m. boundaries. ** p < 0.01, *** p <0.001 by 1-way ANOVA (effect of time).

Figure 2

Figure 2. Circadian rhythms of peroxiredoxin (PRX) oxidation are not affected by transcriptional and translational inhibition

RBCs were entrained under temperature cycles and then kept under constant conditions (at 37°C, in total darkness) and sampled every 4 hours. Representative immunoblots showing oxidised/hyperoxidised peroxiredoxin (PRX-SO2/3) dimer are shown for samples incubated with a, α-amanitin (α-AMN), or b, cycloheximide (CHX) for the entirety of the experiments. Quantification by densitometry is shown below. Values were normalised to the maximum for each blot. Each point represents a mean normalised intensity. n.s., not significant. Further details are shown in Supplementary Fig. 3.

Figure 3

Figure 3. Temperature-compensation of circadian peroxiredoxin oxidation rhythms

Red blood cells were entrained in temperature cycles (12 h at 32°C, 12 h at 37°C) for two complete cycles and then kept under a constant temperature of either 32°C or 37°C for the rest of the experiment and sampled every 4 hours as before. Immunoblots for oxidised/hyperoxidised peroxiredoxin (PRX-SO2/3) dimer obtained from red blood cells from subjects A, B and C are shown. Loading controls (Coomassie-stained gels showing haemoglobin monomer bands) for each blot are also shown. Quantification of the above immunoblots by densitometry is shown on the left of the figure.

Figure 4

Figure 4. Expression patterns and oligomerisation of peroxiredoxins

a, Immunoblots showing expression of the human peroxiredoxin paralogues (PRX1-6) in red blood cells (RBC) and in mouse NIH3T3 cells. Loading of each lane was approximately equal. b, Oligomerisation patterns of PRX and PRX-SO2/3 in red blood cells. Following two cycles of temperature entrainment, cells were kept under constant temperature (37°C) for the rest of the experiment, and sampled every 4 hours. Representative immunoblots for PRX2 and PRX-SO2/3 are shown. Whole blot images in Supplementary Fig. 5 illustrate the different oligomeric forms. Immunoblots were quantified by densitometry for c, PRX-SO2/3 and d, PRX2. Arrowheads indicate peaks of abundance.

Figure 5

Figure 5. Circadian rhythms in haemoglobin oxidation and red blood cell (RBC) metabolism

a, Intrinsic front-face fluorescence (FFF) measurements of RBCs and controls. Experiments performed under constant conditions (at 37°C, in total darkness). Mean values for each time-point are shown (individual traces and further details are in Supplementary Fig. 6a). 2-way ANOVA (group × time) p < 0.001 (***). b, NADH and NADPH concentrations in red blood cells. Mean values (± s.e.m.) for three experimental subjects are shown. 1-way ANOVA (effect of time) for NADH/NADPH data was significant (*** p < 0.001). 2-way ANOVA (metabolite × time) did not reveal a significant difference between NADH and NADPH profiles (n.s., not significant). Individual profiles shown in Supplementary Fig. 6b,c.

Figure 6

Figure 6. Peroxiredoxin rhythms in nucleated cells

a, Peroxiredoxin rhythms in mouse NIH3T3 fibroblasts synchronised by a serum-shock. Immunoblots for Prx1, Prx6 and Prx-SO2/3 dimer are shown, in addition to Bmal1 and a β-actin loading control. b,c Peroxiredoxin rhythms in mouse embryonic fibroblasts (MEFs). MEFs from wild-type or mCry1/2 double-knockout mice were entrained in temperature cycles and then kept under constant temperature (37°C) for the rest of the experiment (as shown in the schematic). b, Representative immunoblots of oxidised/hyperoxidised peroxiredoxin (Prx-SO2/3) dimer. c, Quantification of Prx-SO2/3 immunoblots by densitometry. Mean values (± s.e.m.) for _n_=4 biological replicates are shown.

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