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 a-and b-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 F 1 F 0 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 F 1 F 0 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.

Optical nanosensors are based on particles with diameters from 20 to 200 nm containing sensory elements. The latter are comprised of one or more signaling molecules and one or more references, which allow measurements to be ratiometric and hence independent on the amount of sensor. The signaling molecules may range from simple ion-binding fluorophores, e.g., pH-sensitive dyes, to complex biochemical assays. Aptamers are ideal for use in nanosensors because they are relatively easy to modify chemically and hence to transform into signaling molecules, and their binding affinities may be fine-tuned to a desired measuring range in the selection process. Here we first describe the selection of metabolite binding aptamers, how they are transformed into signaling molecules using a molecular beacon construct and then how they are inserted into nanoparticles. Finally, we briefly describe how the sensors are calibrated before inserted into cells to measure metabolite concentration in real time. As examples we present aptamers binding to key metabolites in cells: ATP and fructose 1, 6-bisphosphate (FBP).

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