On the coupling of intracellular K + ${{\rm{K}}}

We have investigated the interplay between glycolytic oscillations and intracellular K + ${{\rm{K}}}^{+}$ concentration in the yeast Saccharomyces cerevisiae. Intracellular K + ${{\rm{K}}}^{+}$ concentration was measured using the fluorophore potassium-binding benzofuranisophthalate (PBFI). We found that K + ${{\rm{K}}}^{+}$ is an essential ion for the occurrence of glycolytic oscillations and that intracellular K + ${{\rm{K}}}^{+}$ concentration oscillates synchronously with other variables such as nicotinamide adenine dinucleotide hydride (NADH), intracellular adenosine triphosphate (ATP), and mitochondrial membrane potential. We also investigated if glycolysis and intracellular K + ${{\rm{K}}}^{+}$ concentration oscillate in a number of yeast strains with mutations in K + ${{\rm{K}}}^{+}$ transporters in the plasma membrane, mitochondrial membrane and in the vacuolar membrane. Most of these strains are still capable of showing glycolytic oscillations, but two strains are not: (i) a strain with a deletion in the mitochondrial Mdm38p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ transporter and (ii) a strain with deletion of the late endosomal Nhx1p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ( Na + ∕ H + ${\text{Na}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ) transporter. In these two mutant strains intracellular K + ${{\rm{K}}}^{+}$ concentration seems to be low, indicating that the two transporters may be involved in transport of K + ${{\rm{K}}}^{+}$ into the cytosol. In the strain, Mdm38p Δ ${\rm{\Delta }}$ oscillations in glycolysis could be restored by addition of the K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ exchange ionophore nigericin. Furthermore, in two nonoscillating mutant strains with a defective V-ATPase and deletion of the Arp1p protein the intracellular K + ${{\rm{K}}}^{+}$ is relatively high, suggesting that the V-ATPase is essential for transport of K + ${{\rm{K}}}^{+}$ out of the cytosol and that the cytoskeleton may be involved in binding K + ${{\rm{K}}}^{+}$ to reduce the concentration of free ion in the cytosol. Analyses of the time series of oscillations of NADH, ATP, mitochondrial membrane potential, and potassium concentration using data-driven modeling corroborate the conjecture that K + ${{\rm{K}}}^{+}$ ion is essential for the emergence of oscillations and support the experimental findings using mutant strains.

Chaos in the peroxidase-oxidase oscillator.

The peroxidase-oxidase (PO) reaction involves the oxidation of reduced nicotinamide adenine dinucleotide by molecular oxygen. When both reactants are supplied continuously to a reaction mixture containing the enzyme and a phenolic compound, the reaction will exhibit oscillatory behavior. In fact, the reaction exhibits a zoo of dynamical behaviors ranging from simple periodic oscillations to period-doubled and mixed mode oscillations to quasiperiodicity and chaos. The routes to chaos involve period-doubling, period-adding, and torus bifurcations. The dynamic behaviors in the experimental system can be simulated by detailed semiquantitative models. Previous models of the reaction have omitted the phenolic compound from the reaction scheme. In the current paper, we present new experimental results with the oscillating PO reaction that add to our understanding of its rich dynamics, and we describe a new variant of a previous model, which includes the chemistry of the phenol in the reaction mechanism. This new model can simulate most of the experimental behaviors of the experimental system including the new observations presented here. For example, the model reproduces the two main routes to chaos observed in experiments: (i) a period-doubling scenario, which takes place at low pH, and a period-adding scenario involving mixed mode oscillations (MMOs), which occurs at high pH. Our simulations suggest alternative explanations for the pH-sensitivity of the dynamics. We show that the MMO domains are separated by narrow parameter regions of chaotic behavior or quasiperiodicity. These regions start as tongues of secondary quasiperiodicity and develop into strange attractors through torus breakdown.

Functional imaging of a model unicell: Spironucleus vortens as an anaerobic but aerotolerant flagellated protist.

Advances in optical microscopy are continually narrowing the chasm in our appreciation of biological organization between the molecular and cellular levels, but many practical problems are still limiting. Observation is always limited by the rapid dynamics of ultrastructural modifications of intracellular components, and often by cell motility: imaging of the unicellular protist parasite of ornamental fish, Spironucleus vortens, has proved challenging. Autofluorescence of nicotinamide nucleotides and flavins in the 400-580 nm region of the visible spectrum, is the most useful indicator of cellular redox state and hence vitality. Fluorophores emitting in the red or near-infrared (i.e., phosphors) are less damaging and more penetrative than many routinely employed fluors. Mountants containing free radical scavengers minimize fluorophore photobleaching. Two-photon excitation provides a small focal spot, increased penetration, minimizes photon scattering and enables extended observations. Use of quantum dots clarifies the competition between endosomal uptake and exosomal extrusion. Rapid motility (161 µm/s) of the organism makes high resolution of ultrastructure difficult even at high scan speeds. Use of voltage-sensitive dyes determining transmembrane potentials of plasma membrane and hydrogenosomes (modified mitochondria) is also hindered by intracellular motion and controlled anesthesia perturbs membrane organization. Specificity of luminophore binding is always questionable; e.g. cationic lipophilic species widely used to measure membrane potentials also enter membrane-bounded neutral lipid droplet-filled organelles. This appears to be the case in S. vortens, where Coherent Anti-Stokes Raman Scattering (CARS) micro-spectroscopy unequivocally images the latter and simultaneous provides spectral identification at 2840 cm-1. Secondary Harmonic Generation highlights the highly ordered structure of the flagella.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.

An experimental study of the regulation of glycolytic oscillations in yeast.

We have studied oscillating glycolysis in the strain BY4743 and isogenic strains with deletions of genes encoding enzymes in glycolysis, mitochondrial electron transport and ATP synthesis. We found that deletion of the gene encoding the hexokinase 1 isoform does not affect the oscillations while deletion of the gene encoding the hexokinase 2 isoform results in oscillations with smaller amplitude. The latter is associated with an almost 50% decrease in hexokinase activity. Deletions in the genes encoding the α- and ß-subunits of phosphofructokinase abolish the oscillations entirely. This loss in oscillatory activity is associated with a fourfold decrease in phosphofructokinase activity. Deletions of genes encoding subunits of the F1F0 ATPase also inhibit the oscillations in accordance with earlier studies using for example inhibitors. Finally, we identified an apparently new control point involving the mitochondrial cytochrome c oxidase. The latter is difficult to explain as oscillatory activity entails 100% inhibition of this enzyme. The mitochondria of this strain seem to have normal F1F0 ATPase activity. Overall these results support earlier experimental and model studies suggesting that in addition to processes within glycolysis also processes outside this pathway contribute to the control of the oscillatory behaviour.

Nanoparticle embedded enzymes for improved lateral flow sensors.

In this study, combining the nanoparticle embedded sensors with lateral flow assays, a novel strategy for ensuring the quality of signalling in lateral flow assays (LFAs) was developed. A LFA for reactive oxygen species (ROS) is reported that is based on horse radish peroxidase (HRP) which is co-entrapped with Texas Red dextran inside porous polyacrylamide nanoparticles. In this system, enzymes are protected in the porous matrix of polyacrylamide which freely allows the diffusion of the analyte. The sensor is rapid and sensitive for quantification of hydrogen peroxide concentrations. A test solution of hydrogen peroxides was quantified with this novel LFA-ROS sensor to obtain a linear range between 1 and 25 µM. Nanoparticle embedding of enzymes is proposed here as a general strategy for developing enzyme-based lateral flow assays, eliminating adverse effects associated with biological samples.

Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases.

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.

Human myeloperoxidase catalyzes an oscillating peroxidase-oxidase reaction.

We have studied the peroxidase-oxidase reaction catalyzed by human myeloperoxidase in an open system where both substrates-molecular oxygen and NADH-are supplied continuously to the reaction mixture. The reaction shows oscillatory kinetics at pH values around 5, provided that the reaction medium in addition to the enzyme and the substrates also contains an aromatic electron mediator such as melatonin or 4-hydroxybenzoic acid and chloride ions at concentrations >1mM. The experimental findings can be simulated by a detailed model of the reaction. The results are important for our understanding of oxidant production in neutrophils.

Mechanism of melatonin-induced oscillations in the peroxidase-oxidase reaction.

Melatonin induces oscillations in the peroxidase-oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.