Live Cell Imaging Reveals pH Oscillations in Saccharomyces cerevisiae During Metabolic Transitions

Abstract

Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent to which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences of oscillations. In this paper, we report sustained glycolytic oscillations in single cells without the need for cyanide. Glucose addition to immobilized cells induced pH oscillations that could be imaged with fluorescent sensors. A population of cells had oscillations that were heterogeneous in frequency, start time, stop time, duration and amplitude. These changes in cytoplasmic pH were necessary and sufficient to drive changes in NADH. Oscillators had lower mitochondrial membrane potentials and budded more slowly than non-oscillators. We also uncovered a new type of oscillation during recovery from H2O2 challenge. Our data show that pH in S. cerevisiae changes over several time scales, and that imaging pH offers a new way to measure glycolytic oscillations on individual cells.

Modeling studies of heterogeneities in glycolytic oscillations in HeLa cervical cancer cells

“…Heterogeneity in glycolytic oscillations has been reported in yeast cells immobilized on a coverslip, agarose pads, or in a micro uidic chamber (Weber et al, 2012;Dodd and Kralj, 2017;and Gustavsson et al, 2012). However, the degree of the heterogeneity is di erent depending on the strains and experimental conditions.…”

“…S. cerevisiae (strain BY4741) is reported to oscillate very heterogeneous in period (ca. 29-63 s), start time, stop time, duration, and amplitude on agarose pads (Dodd and Kralj, 2017). The reasons for these heterogeneities in yeast glycolytic oscillations are currently unknown; nevertheless, in the rst case, S. cerevisiae (strain X2180), homogeneous distributions in the periods of oscillations are explained by small variations (±2%) in the maximum velocity (V max ) of glucose uptake, which is reported to be su cient to simulate the most (96.2%) of the experimental results (Gustavsson et al, 2012).…”

“…Another characteristics in the present model is that the concentration of glucose in the extracellular solution, G ex , was taken into account. This variable is indispensable for reproducing the in vitro experiments performed in our previous study and yeast glycolytic oscillations (Weber et al, 2012;Amemiya et al, 2015;Dodd and Kralj, 2017;and Shibata et al, 2018). In such experiments, glucose is introduced, at a certain time for few seconds, into a chamber or a vessel in which glucose-starved cells are immobilized or suspended.…”

In vivo characterisation of fluorescent proteins in budding yeast

“…Therefore, the performance of an FP in a pH dynamic or low-pH environment should be assessed in vivo and not in vitro before performing experiments to rule out any pH bias. In S. cerevisiae, pH is tightly controlled but very dynamic and even implied as second messenger (Dodd and Kralj, 2017;Orij et al, 2012Orij et al, , 2009, and so experiments that affect the physiology of yeast should preferably be studied with pH-insensitive FPs. Which factors specifically affect pH sensitivity in vivo is not known, although it is known that ions such as Claffect the pH curves (Griesbeck et al, 2001).…”

Is a constant low-entropy process at the root of glycolytic oscillations?

“…An important and generally overlooked observation, however, is that it is not only a few cellular metabolites (e.g., NADH, ATP) that oscillate during glycolysis, but a large number of cellular properties including intracellular pH, electrical potentials, intracellular water dynamics, and heat [8][9][10][11][12], in ways that strongly suggest that general principles of coupling are at play and that glycolysis as an enzymatic pathway both influences and is influenced by the rest of the cell. This coupling of glycolysis with a host of other cellular phenomena is not adequately described under the assumptions of a largely isolated system of reactions occurring under conditions akin to a dilute solution.…”