Temporal segregation of biosynthetic processes is responsible for metabolic oscillations during the budding yeast cell cycle

Many cell biological and biochemical mechanisms controlling the fundamental process of eukaryotic cell division have been identified; however, the temporal dynamics of biosynthetic processes during the cell division cycle are still elusive. Here, we show that key biosynthetic processes are temporally segregated along the cell cycle. Using budding yeast as a model and single-cell methods to dynamically measure metabolic activity, we observe two peaks in protein synthesis, in the G1 and S/G2/M phase, whereas lipid and polysaccharide synthesis peaks only once, during the S/G2/M phase. Integrating the inferred biosynthetic rates into a thermodynamic-stoichiometric metabolic model, we find that this temporal segregation in biosynthetic processes causes flux changes in primary metabolism, with an acceleration of glucose-uptake flux in G1 and phase-shifted oscillations of oxygen and carbon dioxide exchanges. Through experimental validation of the model predictions, we demonstrate that primary metabolism oscillates with cell-cycle periodicity to satisfy the changing demands of biosynthetic processes exhibiting unexpected dynamics during the cell cycle.

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Sample size
All experiments performed in this study are microscopy-based experiments. In experiments with dynamic addition of inhibitors, auxin or glucose analogue and in subsequent single-cell analyses, we traced the maximal number of cells cultivated in the microfluidic device, with these cells reliably segmented and having smooth trajectories of studied variables according to visual inspection. We consider the sample sizes sufficient to draw reliable conclusions as we observed the reported average patterns in replicate experiments and in experiments using alternative methods. In experiments with constant growth conditions and in subsequent single-cell analyses, we traced either the maximal number of cells in the microfluidic device or a smaller than maximal number of cells, which was enough to obtain reliable average patterns such that these patterns reflect individual single-cell trajectories. For the experiments with the oxygen-level perturbation and with cultivation of carbohydrate-metabolism mutant in constant growth conditions, we demonstrated the dynamics of studied variables in the continuous trajectories of several representative cells as it was sufficient to draw respective conclusions. We indicate the exact number of analyzed cells for each experiment.

Data exclusions We performed visual inspection of cell segmentation and cell tracking quality both on the level of raw time-lapse microscopy images and on
the level of single-cell trajectories of cell volume and fluorescence. We identified obvious artefacts generated by wrong cell segmentation, tracking and focus shifts, which result in abrupt jumps in measured mother or daughter cell volumes and fluorescence. We discarded singlecell trajectories or individual data points in single-cell trajectories affected by such artefacts. In a fraction of cells, we were not able to reliably detect the timing of mitotic exit and START due to noisy Whi5 signal, thus, we did not use the data of those cells in analyses requiring the timing of the cell-cycle events. We also excluded data from cells with extreme durations of the cell cycle or parts of the cell cycle (described in detail in Methods). These data-exclusion criteria were preestablished. The data analysis code (namely, Jupyter Notebooks) that we made available via dataverse.nl (https://doi.org/10.34894/XPYC7Y) documents all data exclusion cases.

Replication
We replicated the following perturbation experiments twice: cycloheximide-based stop-and-respond experiments, cerulenin-based stop-andrespond experiments, Ugp1-depeltion stop-and-respond experiments, glucose-analogue-uptake experiments. The control experiment for the Ugp1-depeltion stop-and-respond experiments was performed once. The experiment to trace the production rate of tetO7-controlled sfGFP during the cell cycle was repeated three times. We present the cell-cycle-resolved dynamics of cell volume and cell surface from one experiment as growth conditions were not perturbed in this experiment, and there is low variability in the dynamics of individual cell-cycle traces. The experiment with the glycolytic-flux biosensor was performed once as growth conditions were not perturbed in this experiment, the finding was reproduced with an alternative method (glucose analogue uptake), and a control experiment without the glycolytic-fluxsensing moiety of the sensor was carried out. The oxygen-level perturbation experiment was performed once as it was sufficient to draw the respective conclusion. The experiments to observe NAD(P)H oscillations on YPD medium and glucose minimal media containing complete

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supplement mixture and lipid mixture were performed once. The experiments to observe NAD(P)H oscillations on glucose minimal medium without other carbon-source supplements and on pyruvate minimal medium were performed twice. Critical findings were reproduced using alternative experimental methods. Replicate cell-cycle-resolved patterns of protein, lipid and polysaccharide synthesis were used as an input of the cell-mass model and metabolic model. Randomization Randomization was not applied since we did not expect the influence of the order of the experiments on their outcome, as judged by our previous experience and publications in the field. Experiments with yeast cells were performed under controlled conditions and with one genetic background.