Emily Kruchek

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.

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.

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.

Diseases such as osteoarthritis (OA) are commonly characterized at the molecular scale by gene expression and subsequent protein production; likewise, the effects of pharmaceutical interventions are typically characterized by the effects of molecular interactions. However, these phenomena are usually preceded by numerous precursor steps, many of which involve significant ion influx or efflux. As a consequence, rapid assessment of cell electrophysiology could play a significant role in unravelling the mechanisms underlying drug interactions and progression of diseases, such as OA. In this study, we used dielectrophoresis (DEP), a technique that allows rapid, label-free determination of the dielectric parameters to assess the role of potassium ions on the dielectric characteristics of chondrocytes, and to investigate the electrophysiological differences between healthy chondrocytes and those from an in vitro arthritic disease model. Our results showed that DEP was able to detect a significant decrease in membrane conductance (6191 ± 738 vs. 8571 ± 1010 S/m2), membrane capacitance (10.3 ± 1.47 vs. 14.5 ± 0.01 mF/m2), and whole cell capacitance (5.4 ± 0.7 vs. 7.5 ± 0.3 pF) following inhibition of potassium channels using 10 mM tetraethyl ammonium, compared to untreated healthy chondrocytes. Moreover, cells from the OA model had a different response to DEP force in comparison to healthy cells; this was seen in terms of both a decreased membrane conductivity (782 S/m2 vs. 1139 S/m2) and a higher whole cell capacitance (9.58 ± 3.4 vs. 3.7 ± 1.3 pF). The results show that DEP offers a high throughput method, capable of detecting changes in membrane electrophysiological properties and differences between disease states.

Even in nonexcitable cells, the membrane potential Vm is fundamental to cell function, with roles from ion channel regulation, development, to cancer metastasis. Vm arises from transmembrane ion concentration gradients; standard models assume homogeneous extracellular and intracellular ion concentrations, and that Vm only exists across the cell membrane and has no significance beyond it. Using red blood cells, we show that this is incorrect, or at least incomplete; Vm is detectable in the extracellular ion concentration beyond the cell surface, and that modulating Vm produces quantifiable and consistent changes in extracellular potential. Evidence strongly suggests this is due to capacitive coupling between Vm and the electrical double layer, rather than molecular transporters. We show that modulating Vm changing the extracellular ion composition mimics the behaviour of voltage-activated ion channel in non-excitable channels. We also observe Vm-synchronised circadian rhythms in extracellular potential, with significant implications for cell-cell interactions and cardiovascular disease.