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.


METHODS
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.
Laxman
Sunil
S
Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
Sutter
Benjamin M
BM
Tu
Benjamin P
BP
D002453:Cell Cycle; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D005947:Glucose; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein