Behavior of a Metabolic Cycling Population at the Single Cell Level as Visualized by Fluorescent Gene Expression Reporters

BACKGROUND: During continuous growth in specific chemostat cultures, budding yeast undergo robust oscillations in oxygen consumption that are accompanied by highly periodic changes in transcript abundance of a majority of genes, in a phenomenon called the Yeast Metabolic Cycle (YMC). This study uses fluorescent reporters of genes specific to different YMC phases in order to visualize this phenomenon and understand the temporal regulation of gene expression at the level of individual cells within the cycling population. METHODOLOGY: Fluorescent gene expression reporters for different phases of the YMC were constructed and stably integrated into the yeast genome. Subsequently, these reporter-expressing yeast were used to visualize YMC dynamics at the individual cell level in cultures grown in a chemostat or in a microfluidics platform under varying glucose concentrations, using fluorescence microscopy and quantitative Western blots. CONCLUSIONS: The behavior of single cells within a metabolic cycling population was visualized using phase-specific fluorescent reporters. The reporters largely recapitulated genome-specified mRNA expression profiles. A significant fraction of the cell population appeared to exhibit basal expression of the reporters, supporting the hypothesis that there are at least two distinct subpopulations of cells within the cycling population. Although approximately half of the cycling population initiated cell division in each permissive window of the YMC, metabolic synchrony of the population was maintained. Using a microfluidics platform we observed that low glucose concentrations appear to be necessary for metabolic cycling. Lastly, we propose that there is a temporal window in the oxidative growth phase of the YMC where the cycling population segregates into at least two subpopulations, one which will enter the cell cycle and one which does not

Redox cycling of intracellular thiols: State variables for ultradian, cell division cycle and circadian cycles?

Since the pioneering work of Rapkine (1931) the hypothesis that many rhythmic life processes may involve cyclic interconversion of dithiols to disulphides provides a central theme that unifies ultradian, cell division cycle and circadian rhythm research. We have shown that in an autonomously-oscillating continuous culture of Saccharomyces cerevisiae ultradian (τ = 40 min) cycles between high and low respiratory states are accompanied by redox cycling of nicotinamide nucleotide(s) and glutathione. This system may provide insights into regulation of rhythmic processes with longer periods