Abstract
The yeast metabolic cycle (YMC) is a fascinating example of biological organization, in which cells constrain the function of specific genetic, protein and metabolic networks to precise temporal windows as they grow and divide. However, understanding the intracellular origins of the YMC remains a challenging goal, as measuring the oxygen oscillations traditionally associated with it requires the use of synchronized cultures growing in nutrient-limited chemostat environments. To address these limitations, we used custom-built microfluidic devices and time-lapse fluorescence microscopy to search for metabolic cycling in the form of endogenous flavin fluorescence in unsynchronized single yeast cells. We uncovered robust and pervasive metabolic cycles that were synchronized with the cell division cycle (CDC) and oscillated across four different nutrient conditions. We then studied the response of these metabolic cycles to chemical and genetic perturbations, showing that their phase synchronization with the CDC can be altered through treatment with rapamycin, and that metabolic cycles continue even in respiratory deficient strains. These results provide a foundation for future studies of the physiological importance of metabolic cycles in processes such as CDC control, metabolic regulation and cell aging.
Highlights
Oscillations underlie a wide variety of biological phenomena
Our results indicate that the metabolic cycle is a robust oscillator that typically couples one-to-one to the cell division cycle across four different nutrient conditions, we found that treatment with rapamycin can alter phase synchrony between these two oscillators, leading to multiple metabolic cycles per CDC and increased variability of metabolic cycling
We reasoned that we could utilize flavin molecules as dynamic reporters of metabolic cycling in single cells because of their unique fluorescent properties; they fluoresce in the visible spectrum[31] and their reduced equivalents (FADH2 and FMNH2) have negligible fluorescence in this range[32], allowing their oxidized and reduced states to be distinguished
Summary
Oscillations underlie a wide variety of biological phenomena. Their unique dynamical characteristics allow organisms across diverse kingdoms of life and at multiple length scales to perform a myriad of complex functions such as timekeeping[1], resource allocation and sharing[2], as well as coordinated behavior[3]. Despite the importance of these findings, the extent to which the many features of the YMC are recapitulated at the single-cell level remains to be determined Answering these questions is made all the more difficult by the fact that different experimental set-ups can lead to markedly different observations about the period of the metabolic cycle and its relationship to the cell division cycle. In contrast to the chemostat studies of the YMC, we found that cellular respiration is dispensable for metabolic cycling, as flavin oscillations persisted in deletion mutants impaired in oxidative phosphorylation These results are in general agreement with the previous findings from Papagiannakis et al.[12], and demonstrate that another critical cellular metabolite, flavin, oscillates during the cell division cycle. Our work provides new insights into metabolic cycling in budding yeast and provides a foundation and methodology for future studies of its role at the level of single cells
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