Single-cell study links metabolism with nutrient signaling and reveals sources of variability

Abstract

Background: The yeast AMPK/SNF1 pathway is best known for its role in glucose de/repression. When glucose becomes limited, the Snf1 kinase is activated and phosphorylates the transcriptional repressor Mig1, which is then exported from the nucleus. The exact mechanism how the Snf1-Mig1 pathway is regulated is not entirely elucidated.

Results: Glucose uptake through the low affinity transporter Hxt1 results in nuclear accumulation of Mig1 in response to all glucose concentrations upshift, however with increasing glucose concentration the nuclear localization of Mig1 is more intense. Strains expressing Hxt7 display a constant response to all glucose concentration upshifts. We show that differences in amount of hexose transporter molecules in the cell could cause cell-to-cell variability in the Mig1-Snf1 system. We further apply mathematical modelling to our data, both general deterministic and a nonlinear mixed effect model. Our model suggests a presently unrecognized regulatory step of the Snf1-Mig1 pathway at the level of Mig1 dephosphorylation. Model predictions point to parameters involved in the transport of Mig1 in and out of the nucleus as a majorsource of cell to cell variability.

Conclusions: With this modelling approach we have been able to suggest steps that contribute to the cell-to-cell variability. Our data indicate a close link between the glucose uptake rate, which determines the glycolytic rate, and the activity of the Snf1/Mig1 system. This study hence establishes a close relation between metabolism and signalling.

Keywords: Dynamical modelling; Glucose uptake; Microfluidics systems; Non-linear mixed effect modelling.

Fig. 1

Localization of Mig1 over time expressed as nuclear fluorescence intensity divided by the cytosolic fluorescence intensity. Each graph depicts the results of one experiment. The y axis is distributed logarithmically. Each grey line represents the trace of one single cell and the average of all cells is represented with a thicker blue line. Between 22 to 41 cells were analysed in each experiment. The different strains are displayed vertically (wild type (WT) (a), HXT1 (b), HXT7 (c)) and the different concentrations are displayed horizontally

Fig. 2

Study of the cell-to-cell variability observed in the Snf1/Mig1 system. (a)(b) Hxt7-GFP before and following a switch from ethanol media to media containing 220 mM glucose. (a) Time lapse microscopic images, upper images show HXT7-GFP, the lower images show brightfield. (b) Fluorescence intensity along an intersection through the yeast cells. The fluorescence intensity is higher at the points the intersection line crosses the cell membrane and does not change over time. The result of only one cell is displayed but multiple cells were analyzed and none of the cells showed a decrease in membrane localization of the Hxt7 transporter after 15 min following the shift to glucose media. (c) The ratio 15 min after glucose upshift plotted over the cell size for the HXT1 strain. The cell size is plotted on the x-axes. As a measurement for the Mig1-Snf1 pathway response we chose the Mig1 nuclear/cytosolic ratio. Upshifts to higher glucose concentration; 0 to 220 mM (blue diamonds), 0 to 55 mM (red squares) and 0 to 27.5 mM glucose (green triangle) result in higher final ratio while upshifts to lower glucose concentration 0 to 11 mM glucose (purple crosses), 0 to 2.75 mM (blue stars) and 0 to 0 mM glucose (orange dots) display a lower final ratio. (d) Hxt7-GFP pregrown overnight in 3% ethanol media. Upper image shows the bright field image, lower image shows the cellular distribution of Hxt7-GFP

Fig. 3

Dynamic and NLME modelling of the Snf1/Mig1 pathway. a Schematic representation of the model. The model consists of three main parts, namely the activity of glucose, the activity of Snf1 and the activity of Mig1. b Simulation of the distribution of the random parameters for the HXT1 strain. The columns indicate the extracellular glucose concentration, ranging from 0, 11, 55 to 220 mM which are illustrated from the left to the right in the figure. Each heat map is generated by drawing 50 mixed effect random terms, that is η ∼ N(0, σ), corresponding to the parameter vectors from the generated parameter distributions for the various strains and glucose concentrations. The heat map displays various parameters on the y-axis, the individuals on the x-axis and the magnitude of the random terms are indicated by the colour scale shown above the figure. The colour scale ranges from 0 to 2 where a red colour corresponds to a high random term and a blue colour correspond to a low value of the random term. The white fields correspond to the parameters connected to the hexose transporters that are not active in HXT1 strains