Circadian rhythmicity in murine blood:Electrical effects of malaria infection and anemia

Circadian rhythms are biological adaptations to the day-night cycle, whereby cells adapt to changes in the external environment or internal physiology according to the time of day. Whilst many cellular clock mechanisms involve gene expression feedback mechanisms, clocks operate even where gene expression is absent. For example, red blood cells (RBCs) do not have capacity for gene expression, and instead possess an electrophysiological oscillator where cytosolic potassium plays a key role in timekeeping. We examined murine blood under normal conditions as well as in two perturbed states, malaria infection and induced anemia, to assess changes in baseline cellular electrophysiology and its implications for the electrophysiological oscillator. Blood samples were analyzed at 4-h intervals over 2 days by dielectrophoresis, and microscopic determination of parasitemia. We found that cytoplasmic conductivity (indicating the concentration of free ions in the cytoplasm and related to the membrane potential) exhibited circadian rhythmic behavior in all three cases (control, malaria and anemia). Compared to control samples, cytoplasm conductivity was decreased in the anemia group, whilst malaria-infected samples were in antiphase to control. Furthermore, we identified rhythmic behavior in membrane capacitance of malaria infected cells that was not replicated in the other samples. Finally, we reveal the historically famous rhythmicity of malaria parasite replication is in phase with cytoplasm conductivity. Our findings suggest the electrophysiological oscillator can impact on malaria parasite replication and/or is vulnerable to perturbation by rhythmic parasite activities

Casein Kinase 1 Underlies Temperature Compensation of Circadian Rhythms in Human Red Blood Cells

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles

The platelet electrome: evidence for a role in regulation of function and surface interaction

Introduction. Platelets protect the body from injury through formation of blood clots, changing from a normal, quiescent state to become “activated” in response to external stimuli such as chemical cues, shear stress and temperature. This causes changes in shape, increased adhesion, and alteration of the electrical properties such as membrane potential Vm and zeta potential ζ. These phenomena have been regarded as largely unconnected; for example, changes in ζ have been attributed solely to alteration of surface lipid concentration. However, recent reports suggest that cells can alter ζ electrostatically by alteration of Vm in red blood cells. We hypothesised that if platelets also modulate ζ via Vm, this may provide an alternative mechanism to alter cell-cell interaction. Methods. We investigated platelets stored at different temperatures (4°C, 22°C, 37°C) for 24h, which is known to alter platelet behaviour and electrical properties, and compared these with analyses of freshly-harvested platelets. These four conditions exhibited unique sets of electrical properties (Vm, ζ, membrane conductance Geff and cytoplasm conductivity σcyto) as well as surface exposure of the adhesion molecule P-Selectin. These were analysed to identify correlations between electrical parameters and platelet activation state. Results. Many parameters exhibit pairwise correlation across all four conditions, in particular between ζ and Geff, and also between Vm and σcyto. Furthermore, when the electrical behaviour of platelets stored at 4°C (known to activate the cells) was removed from the analysis, additional relationships were observed among the remaining conditions, including those connecting ζ and Vm to the amount of P-selectin binding. Conclusion. Results suggest that Vm may mechanistically alter the availability of cationic molecules at the cell surface, a process never reported before, with implications for our wider understanding of cell-molecule and cell-cell interaction

Casein kinase 1 underlies temperature compensation of circadian rhythms in human red blood cells

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature compensated circadian rhythms which, since RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on casein kinases in RBCs. As with nucleated cell types, broad spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles