Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor (2019)
Figure 7

Figure 7. The glycolytic flux sensor can measure glycolytic flux in individual cells. (A) Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. (B) Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished. (C) Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). (D) YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose. (E) Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium. (F-G) The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. Specifically, we computed the average cell-cycle-relative timing for each of these events ${\overline{\varphi}}^{e}$ in the following way: $\forall e \in E\ {\overline{\varphi}}^{e} = \frac{1}{N}\sum_{cc = 1}^{N}\frac{t_{\text{cc}}^{e} - t_{\text{cc}}^{\text{cyt}}}{t_{\text{cc}}^{\text{next\ cyt}} - t_{\text{cc}}^{\text{cyt}}}$, where N is the number of cell cycles in the replicate of interest, tcce is the time in minutes when the event e happens in the cell cycle cc. The orange vertical lines denote ${\overline{\varphi}}^{\text{budding}}$ for both replicates. In the aligned cell cycles, we converted the time in minutes t to the phase φcc in the following way: $\varphi_{\text{cc}} = ({\overline{\varphi}}^{E\left\lbrack i + 1 \right\rbrack} - {\overline{\varphi}}^{E\lbrack i\rbrack})\frac{t - t_{\text{cc}}^{E\lbrack i\rbrack}}{t_{\text{cc}}^{E\lbrack i + 1\rbrack} - t_{\text{cc}}^{E\lbrack i\rbrack}} + {\overline{\varphi}}^{E\lbrack i\rbrack}$ for t ∈ [tccE[i], tccE[i+1]] if E[i]= cyt or t ∈ (tccE[i], tccE[i+1]] if E[i]≠ cyt, where i is the index number of an event in the array E. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.