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• W2562645716 abstract "•Metabolic cycles are an intrinsic, growth-condition-independent behavior of single cells•The metabolic oscillations are not the result of the cell cycle and thus are autonomous•The metabolic oscillator and the cyclin/CDK machinery form a system of coupled oscillators•Both the early and late cell cycle operate in coordination with the metabolic oscillator Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell-cycle initiation and progression. Using microfluidics, cell-cycle reporters, and single-cell metabolite measurements, we found that metabolism of budding yeast is a CDK-independent oscillator that oscillates across different growth conditions, both in synchrony with and also in the absence of the cell cycle. Using environmental perturbations and dynamic single-protein depletion experiments, we found that the metabolic oscillator and the cell cycle form a system of coupled oscillators, with the metabolic oscillator separately gating and maintaining synchrony with the early and late cell cycle. Establishing metabolism as a dynamic component within the cell-cycle network opens new avenues for cell-cycle research and therapeutic interventions for proliferative disorders. Eukaryotic cell division is known to be controlled by the cyclin/cyclin dependent kinase (CDK) machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell-cycle initiation and progression. Using microfluidics, cell-cycle reporters, and single-cell metabolite measurements, we found that metabolism of budding yeast is a CDK-independent oscillator that oscillates across different growth conditions, both in synchrony with and also in the absence of the cell cycle. Using environmental perturbations and dynamic single-protein depletion experiments, we found that the metabolic oscillator and the cell cycle form a system of coupled oscillators, with the metabolic oscillator separately gating and maintaining synchrony with the early and late cell cycle. Establishing metabolism as a dynamic component within the cell-cycle network opens new avenues for cell-cycle research and therapeutic interventions for proliferative disorders. Initiation and progression of the cell cycle are considered to occur in response to the timely ordered transcriptional, post-transcriptional, and post-translational regulation of the cell cycle (cyclin/cyclin dependent kinase [CDK]) machinery components (Barik et al., 2010Barik D. Baumann W.T. Paul M.R. Novak B. Tyson J.J. A model of yeast cell-cycle regulation based on multisite phosphorylation.Mol. Syst. Biol. 2010; 6: 405Crossref PubMed Scopus (93) Google Scholar, Coudreuse and Nurse, 2010Coudreuse D. Nurse P. Driving the cell cycle with a minimal CDK control network.Nature. 2010; 468: 1074-1079Crossref PubMed Scopus (282) Google Scholar, Tyson and Novak, 2008Tyson J.J. Novak B. Temporal organization of the cell cycle.Curr. Biol. 2008; 18: R759-R768Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, there is evidence that a cell-cycle regulator external to the cyclin/CDK machinery provides triggers for cell-cycle initiation or progression. First, cell-cycle entry can occur even in the absence of major cell-cycle machinery components (e.g., the early cyclins) (Sherr and Roberts, 2004Sherr C.J. Roberts J.M. Living with or without cyclins and cyclin-dependent kinases.Genes Dev. 2004; 18: 2699-2711Crossref PubMed Scopus (908) Google Scholar). Second, late cell-cycle proteins (e.g., cdc14 and sic1) (Lu and Cross, 2010Lu Y. Cross F.R. Periodic cyclin-Cdk activity entrains an autonomous Cdc14 release oscillator.Cell. 2010; 141: 268-279Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Rahi et al., 2016Rahi S.J. Pecani K. Ondracka A. Oikonomou C. Cross F.R. The CDK-APC/C oscillator predominantly entrains periodic cell-cycle transcription.Cell. 2016; 165: 475-487Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and possibly also global transcription (Haase and Reed, 1999Haase S.B. Reed S.I. Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle.Nature. 1999; 401: 394-397Crossref PubMed Scopus (90) Google Scholar, Orlando et al., 2008Orlando D.A. Lin C.Y. Bernard A. Wang J.Y. Socolar J.E.S. Iversen E.S. Hartemink A.J. Haase S.B. Global control of cell-cycle transcription by coupled CDK and network oscillators.Nature. 2008; 453: 944-947Crossref PubMed Scopus (229) Google Scholar), continue to oscillate in cell-cycle-arrested cells. Third, the eukaryotic cell cycle evolved before CDKs, and thus, the early eukaryotes must have employed non-CDK cell-cycle regulators (Krylov et al., 2003Krylov D.M. Nasmyth K. Koonin E.V. Evolution of eukaryotic cell cycle regulation: stepwise addition of regulatory kinases and late advent of the CDKs.Curr. Biol. 2003; 13: 173-177Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Because metabolism oscillates in synchrony with (Brunetti et al., 2016Brunetti A.J. Aydin M. Buchler N.E. Cell cycle Start is coupled to entry into the yeast metabolic cycle across diverse strains and growth rates.Mol Biol Cell. 2016; 27: 64-74Crossref PubMed Scopus (36) Google Scholar, Futcher, 2006Futcher B. Metabolic cycle, cell cycle, and the finishing kick to Start.Genome Biol. 2006; 7: 107Crossref PubMed Scopus (52) Google Scholar, Klevecz et al., 2004Klevecz R.R. Bolen J. Forrest G. Murray D.B. A genomewide oscillation in transcription gates DNA replication and cell cycle.Proc. Natl. Acad. Sci. USA. 2004; 101: 1200-1205Crossref PubMed Scopus (248) Google Scholar, Müller et al., 2003Müller D. Exler S. Aguilera-Vázquez L. Guerrero-Martín E. Reuss M. Cyclic AMP mediates the cell cycle dynamics of energy metabolism in Saccharomyces cerevisiae.Yeast. 2003; 20: 351-367Crossref PubMed Scopus (59) Google Scholar, Silverman et al., 2010Silverman S.J. Petti A.A. Slavov N. Parsons L. Briehof R. Thiberge S.Y. Zenklusen D. Gandhi S.J. Larson D.R. Singer R.H. Botstein D. Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate.Proc. Natl. Acad. Sci. USA. 2010; 107: 6946-6951Crossref PubMed Scopus (68) Google Scholar, Tu et al., 2005Tu B.P. Kudlicki A. Rowicka M. McKnight S.L. Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes.Science. 2005; 310: 1152-1158Crossref PubMed Scopus (691) Google Scholar, Tu et al., 2007Tu B.P. Mohler R.E. Liu J.C. Dombek K.M. Young E.T. Synovec R.E. McKnight S.L. Cyclic changes in metabolic state during the life of a yeast cell.Proc. Natl. Acad. Sci. USA. 2007; 104: 16886-16891Crossref PubMed Scopus (197) Google Scholar, Xu and Tsurugi, 2006Xu Z. Tsurugi K. A potential mechanism of energy-metabolism oscillation in an aerobic chemostat culture of the yeast Saccharomyces cerevisiae.FEBS J. 2006; 273: 1696-1709Crossref PubMed Scopus (39) Google Scholar) and, as suggested, without the cell cycle (Novak et al., 1988Novak B. Halbauer J. Laszlo E. The effect of CO2 on the timing of cell cycle events in fission yeast Schizosaccharomyces pombe.J. Cell Sci. 1988; 89: 433-439Google Scholar, Slavov et al., 2011Slavov N. Macinskas J. Caudy A. Botstein D. Metabolic cycling without cell division cycling in respiring yeast.Proc. Natl. Acad. Sci. USA. 2011; 108: 19090-19095Crossref PubMed Scopus (54) Google Scholar), and because metabolic checkpoints exist in the cell-cycle program (Jones et al., 2005Jones R.G. Plas D.R. Kubek S. Buzzai M. Mu J. Xu Y. Birnbaum M.J. Thompson C.B. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint.Mol. Cell. 2005; 18: 283-293Abstract Full Text Full Text PDF PubMed Scopus (1295) Google Scholar, Saqcena et al., 2013Saqcena M. Menon D. Patel D. Mukhopadhyay S. Chow V. Foster D.A. Amino acids and mTOR mediate distinct metabolic checkpoints in mammalian G1 cell cycle.PLoS ONE. 2013; 8: e74157Crossref PubMed Scopus (57) Google Scholar, Takubo et al., 2013Takubo K. Nagamatsu G. Kobayashi C.I. Nakamura-Ishizu A. Kobayashi H. Ikeda E. Goda N. Rahimi Y. Johnson R.S. Soga T. et al.Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells.Cell Stem Cell. 2013; 12: 49-61Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar), we conjectured that metabolism operates as an autonomous, cell-cycle-independent oscillator, which together with the cell cycle might form a system of coupled oscillators. In response to nutrients, the metabolic oscillator could orbit with different frequencies and provide periodic triggers for cell-cycle initiation and progression. Interactions between metabolites and cell-cycle proteins (Buchakjian and Kornbluth, 2010Buchakjian M.R. Kornbluth S. The engine driving the ship: metabolic steering of cell proliferation and death.Nat. Rev. Mol. Cell Biol. 2010; 11: 715-727Crossref PubMed Scopus (168) Google Scholar, Lee and Finkel, 2013Lee I.H. Finkel T. Metabolic regulation of the cell cycle.Curr. Opin. Cell Biol. 2013; 25: 724-729Crossref PubMed Scopus (41) Google Scholar, Shi and Tu, 2013Shi L. Tu B.P. Acetyl-CoA induces transcription of the key G1 cyclin CLN3 to promote entry into the cell division cycle in Saccharomyces cerevisiae.Proc. Natl. Acad. Sci. USA. 2013; 110: 7318-7323Crossref PubMed Scopus (95) Google Scholar, Yalcin et al., 2014Yalcin A. Clem B.F. Imbert-Fernandez Y. Ozcan S.C. Peker S. O’Neal J. Klarer A.C. Clem A.L. Telang S. Chesney J. 6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27.Cell Death Dis. 2014; 5: e1337Crossref PubMed Scopus (126) Google Scholar) could convey those triggers, and in reverse, the cell cycle could entrain metabolism via the regulation of enzyme activity (Ewald et al., 2016Ewald J.C. Kuehne A. Zamboni N. Skotheim J.M. The yeast cyclin-dependent kinase routes carbon fluxes to fuel cell cycle progression.Mol. Cell. 2016; 62: 532-545Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, Lee et al., 2014Lee Y. Dominy J.E. Choi Y.J. Jurczak M. Tolliday N. Camporez J.P. Chim H. Lim J.-H. Ruan H.-B. Yang X. et al.Cyclin D1-Cdk4 controls glucose metabolism independently of cell cycle progression.Nature. 2014; 510: 547-551Crossref PubMed Scopus (171) Google Scholar, Tudzarova et al., 2011Tudzarova S. Colombo S.L. Stoeber K. Carcamo S. Williams G.H. Moncada S. Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-β-TrCP, sequentially regulate glycolysis during the cell cycle.Proc. Natl. Acad. Sci. USA. 2011; 108: 5278-5283Crossref PubMed Scopus (92) Google Scholar, Wang et al., 2014Wang Z. Fan M. Candas D. Zhang T.-Q. Qin L. Eldridge A. Wachsmann-Hogiu S. Ahmed K.M. Chromy B.A. Nantajit D. et al.Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression.Dev. Cell. 2014; 29: 217-232Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, Zhao et al., 2016Zhao G. Chen Y. Carey L. Futcher B. Cyclin-Dependent Kinase Co-Ordinates Carbohydrate Metabolism and Cell Cycle in S. cerevisiae.Mol. Cell. 2016; 62: 546-557Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Here, using methods for the dynamic quantification of metabolites in single cells, in combination with microfluidics and time-lapse microscopy, we demonstrate that the metabolism of budding yeast is an oscillator, which orbits autonomously of the cell cycle. Perturbation experiments, including dynamic nutrient shifts as well as conditional and targeted depletion of cell-cycle proteins, revealed that the metabolic oscillator, together with the cell cycle, forms a system of coupled oscillators. The metabolic and the cell-cycle oscillators accomplish frequency synchrony—required for the activation and progression of the cell-division program—only within a certain window of metabolic frequencies, whereas the robust gating of the cell-cycle phases by metabolic dynamics ensures the temporal separation of biomass production (early cell cycle) and segregation (late cell cycle). Our findings demonstrate that the metabolic oscillator is an indispensable component of cell-cycle regulation, open new research avenues into cell-cycle control, and suggest the metabolic oscillator as a global therapeutic target against proliferative disorders. To test our hypothesis, after which metabolism is an oscillator that is coupled to the cell cycle, we used Saccharomyces cerevisiae as a model. First, we asked whether a metabolic oscillator exists. Because it has been conjectured that population-level cell-cycle synchronization and cell-to-cell communication artificially induce metabolic oscillations (Aon et al., 2007Aon M.A. Cortassa S. Lemar K.M. Hayes A.J. Lloyd D. Single and cell population respiratory oscillations in yeast: a 2-photon scanning laser microscopy study.FEBS Lett. 2007; 581: 8-14Crossref PubMed Scopus (41) Google Scholar, Laxman et al., 2010Laxman S. Sutter B.M. Tu B.P. Behavior of a metabolic cycling population at the single cell level as visualized by fluorescent gene expression reporters.PLoS ONE. 2010; 5: e12595Crossref PubMed Scopus (19) Google Scholar, Sohn et al., 2000Sohn H.Y. Murray D.B. Kuriyama H. Ultradian oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide mediates population synchrony.Yeast. 2000; 16: 1185-1190Crossref PubMed Scopus (80) Google Scholar), we investigated metabolic and cell-cycle dynamics on the single-cell level. We used a microfluidic device for the long-term microscopic observation of single budding yeast cells (Huberts et al., 2013Huberts D.H.E.W. Sik Lee S. Gonzáles J. Janssens G.E. Vizcarra I.A. Heinemann M. Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast.Nat. Protoc. 2013; 8: 1019-1027Crossref PubMed Scopus (27) Google Scholar), the auto-fluorescence of the reduced nicotinamide nucleotide NAD(P)H, to assess its intracellular levels (Gustavsson et al., 2012Gustavsson A.-K. van Niekerk D.D. Adiels C.B. du Preez F.B. Goksör M. Snoep J.L. Sustained glycolytic oscillations in individual isolated yeast cells.FEBS J. 2012; 279: 2837-2847Crossref PubMed Scopus (58) Google Scholar, Lloyd et al., 2002Lloyd D. Salgado L.E.J. Turner M.P. Suller M.T.E. Murray D. Cycles of mitochondrial energization driven by the ultradian clock in a continuous culture of Saccharomyces cerevisiae.Microbiology. 2002; 148: 3715-3724Crossref PubMed Scopus (58) Google Scholar), and a protein-based Förster resonance energy transfer (FRET) sensor to measure ATP (Imamura et al., 2009Imamura H. Nhat K.P. Togawa H. Saito K. Iino R. Kato-Yamada Y. Nagai T. Noji H. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.Proc. Natl. Acad. Sci. USA. 2009; 106: 15651-15656Crossref PubMed Scopus (706) Google Scholar). Optimization of the sensor expression and imaging settings led to adequate signal intensities with marginal cellular photo-damage and photo-toxicity during long-term (>12 hr) imaging (Figures S1A and S1B). Using these tools, we first investigated whether periodic NAD(P)H and ATP fluctuations occur in single cells, grown on high (10 gL−1) glucose without cell-to-cell communication or cell-cycle synchronization (Figure S1C). These fluctuations, subsequently identified (Figures 1A and 1B ; Movie S1) and confirmed by autocorrelation analysis (Figures S2A and S2B), occurred with an average period of ∼2 hr, which corresponds to the average doubling time under this condition. Through comparative analyses, we validated that the measured single-cell FRET signals reflect intracellular ATP concentrations (Figures S1D–S1H). Because the ATP and NAD(P)H signals oscillate oppositely in phase (Figures S2C and S2D), we conclude that the measured metabolite dynamics are not due to a correlated variability (such as periodic volume chances). Given the absence of cell-cycle synchronization (Figure S1C), our findings demonstrate that, contrary to previous reports (Aon et al., 2007Aon M.A. Cortassa S. Lemar K.M. Hayes A.J. Lloyd D. Single and cell population respiratory oscillations in yeast: a 2-photon scanning laser microscopy study.FEBS Lett. 2007; 581: 8-14Crossref PubMed Scopus (41) Google Scholar, Laxman et al., 2010Laxman S. Sutter B.M. Tu B.P. Behavior of a metabolic cycling population at the single cell level as visualized by fluorescent gene expression reporters.PLoS ONE. 2010; 5: e12595Crossref PubMed Scopus (19) Google Scholar, Sohn et al., 2000Sohn H.Y. Murray D.B. Kuriyama H. Ultradian oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide mediates population synchrony.Yeast. 2000; 16: 1185-1190Crossref PubMed Scopus (80) Google Scholar), metabolic cycles with periods in the hour range are an intrinsic behavior of single cells. To test whether metabolic cycles also occur in other growth conditions, we subjected yeast to different nutrients and metabolic operations (aerobic fermentation, respiration, and gluconeogenesis), which varied the doubling time in single cells from 1.4 to 11 hr. Despite these different metabolic operations, we consistently identified oscillations in the NAD(P)H and ATP levels (Figures S2E–S2J), demonstrating that the metabolic cycles occur regardless of growth conditions. To investigate whether the metabolic cycles occur in synchrony with the cell cycle, we correlated the frequencies of budding with the frequencies of the NAD(P)H or ATP oscillations. Despite the wide range of doubling times across nutrient conditions, the budding frequencies always matched the frequencies of the corresponding NAD(P)H or ATP oscillations (Figure 1C). Thus, the metabolic oscillations and the cell cycle operate in frequency synchrony over the range of growth conditions tested, which suggests a coupling between the two periodic processes. To determine if the metabolic oscillations are a mere consequence of cell-cycle operation or if they occur in a cell-cycle-independent manner and thus are autonomous, we searched in our single-cell data for metabolic oscillations that were unaccompanied by cell-cycle progression. Such events occurred for all growth conditions (Figures 2A, S3A, and S3B), with an approximate incidence of 1/50 metabolic oscillations on 10 gL−1 glucose. On 0.01 gL−1 glucose, we also found many cells with consecutive metabolic oscillations without cell-cycle progression (Figures 2A, S3C, and S3D). To determine the cell-cycle status of the non-dividing cells, we used a strain with fluorescently tagged Whi5, a transcriptional repressor of early cyclins and target of CDK phosphorylation. Whi5 sequesters into the nucleus at late mitosis (hereafter denoted as “M exit”) and exits upon phosphorylation at late G1 (denoted as “START”), reporting an active CDK (Bloom and Cross, 2007Bloom J. Cross F.R. Multiple levels of cyclin specificity in cell-cycle control.Nat. Rev. Mol. Cell Biol. 2007; 8: 149-160Crossref PubMed Scopus (401) Google Scholar, Costanzo et al., 2004Costanzo M. Nishikawa J.L. Tang X. Millman J.S. Schub O. Breitkreuz K. Dewar D. Rupes I. Andrews B. Tyers M. CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast.Cell. 2004; 117: 899-913Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, Ferrezuelo et al., 2012Ferrezuelo F. Colomina N. Palmisano A. Garí E. Gallego C. Csikász-Nagy A. Aldea M. The critical size is set at a single-cell level by growth rate to attain homeostasis and adaptation.Nat. Commun. 2012; 3: 1012Crossref PubMed Scopus (116) Google Scholar) (Figures S4A–S4C). Using this reporter, we found that cells with metabolic oscillations but without an accompanying cell cycle were either arrested at G1 (i.e., Whi5 in the nucleus; Figure 2B; Movie S2) or occasionally after budding in a non-G1 phase (i.e., Whi5 in the cytoplasm; Movie S3). To substantiate the finding that metabolic oscillations are not the consequence of the cell-cycle operation, we added the mating pheromone (alpha factor), which induces G1 arrest (Bardwell, 2004Bardwell L. A walk-through of the yeast mating pheromone response pathway.Peptides. 2004; 25: 1465-1476Crossref PubMed Scopus (113) Google Scholar), to cells growing in the microfluidic device. Also after the pheromone-induced cell-cycle arrest, the NAD(P)H levels continued to oscillate (Figures 2C and S5). Together, these findings demonstrate that the metabolic oscillations are not the result of cell-cycle operation and CDK activity but constitute an autonomous behavior of metabolism, occurring across growth conditions. The autonomy of the metabolic oscillator, and its frequency synchrony with the cell cycle (Figure 1C) in normally dividing cells, suggest metabolism as a separate component in the cell-cycle control engine. We conjectured that the metabolic oscillator and the cell-cycle oscillator form a system of coupled oscillators, similar to other instances of synchrony in biology, including the rhythmic flashes of fireflies or the synchronized discharge of cardiac pacemaker cells (Strogatz, 2001Strogatz S.H. Exploring complex networks.Nature. 2001; 410: 268-276Crossref PubMed Scopus (6337) Google Scholar). Analogously to the fact that an effective contraction of the heart muscle requires a strict synchrony between cells in the sinoatrial node, cell-cycle control could emerge from the coupling and mutual entrainment between the metabolic oscillator and the cell-cycle oscillator. To investigate whether the metabolic oscillator and the cell cycle indeed form a coupled oscillator system, we searched for signature features of such systems by means of steady-state and dynamic perturbations. A common characteristic of coupled oscillators is that their natural frequencies (i.e., the frequency of each individual oscillator when uncoupled) converge to a common compromise frequency (i.e., the common frequency of the oscillators when they are coupled) proportional to the strength of their coupling (Strogatz, 2014Strogatz S.H. Nonlinear Dynamics and Chaos. Westview Press, 2014Google Scholar). We determined the frequency of the metabolic oscillator in the presence of cell cycle (compromise frequency) in normally dividing cells and in the absence of cell-cycle progression (natural frequency of the metabolic oscillator), the latter in cells where the cell cycle was arrested with the alpha factor (Figure S5). In line with the theory of coupled oscillators (Strogatz, 2014Strogatz S.H. Nonlinear Dynamics and Chaos. Westview Press, 2014Google Scholar), we found a linear correlation between the natural metabolic and compromise frequencies under different conditions (Figure 3A). The compromise frequency was routinely 16% lower than its corresponding natural metabolic frequency (Figure 3A), a reduction that could be interpreted as a load imposed on the metabolic oscillator by the cell cycle upon coupling. Another distinctive feature of coupled oscillator systems is that coupling and synchrony are only accomplished when the natural frequencies of the individual oscillators are proximal (Nolte, 2015Nolte D.D. Introduction to Modern Dynamics: Chaos, Networks, Space and Time. Oxford University Press, 2015Google Scholar, Strogatz, 2014Strogatz S.H. Nonlinear Dynamics and Chaos. Westview Press, 2014Google Scholar). With different nutrient conditions resulting in different natural frequencies for the metabolic oscillator (Figure 3A), we investigated at which natural metabolic frequencies coupling with the cell cycle occurs. Here, we found that at very low metabolic frequencies and at very high ones, no coupling occurred (Figure 3B). Given the standardized reduction of the compromise frequency (16%) (Figure 3A), we identified the range of natural metabolic frequencies from 0.15 h−1 to 0.96 h−1 to enable coupling (Figure 3B). On 0.01 gL−1 glucose, 75% of the metabolic oscillations without cell cycle (Figures S3C and S3D) had a natural metabolic frequency below the estimated threshold for coupling and thus cell-cycle initiation (Figure 3B), resulting in a substantial fraction of non-dividing cells. A statistical analysis (hazard function analysis; Supplemental Experimental Procedures) of the dynamic NAD(P)H signals and START occurrence further demonstrates the importance of the metabolic frequency for coupling and cell-cycle initiation (Figure 3C). These findings reveal a second characteristic of coupled oscillators: coupling between the metabolic oscillator and the cell cycle is only achieved within a window of natural metabolic frequencies; lower or higher metabolic frequencies are not sufficient for coupling and thus for cell-cycle initiation. Another characteristic of coupled oscillators is phase gating, i.e., the maintenance of a relative phase between oscillators in synchrony (Feillet et al., 2014Feillet C. Krusche P. Tamanini F. Janssens R.C. Downey M.J. Martin P. Teboul M. Saito S. Lévi F.A. Bretschneider T. et al.Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.Proc. Natl. Acad. Sci. USA. 2014; 111: 9828-9833Crossref PubMed Scopus (141) Google Scholar, Mori et al., 1996Mori T. Binder B. Johnson C.H. Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours.Proc. Natl. Acad. Sci. USA. 1996; 93: 10183-10188Crossref PubMed Scopus (253) Google Scholar). Initially focusing on cells grown on high (10 gL−1) glucose, we found strict phase gating of the cell-cycle events at specific metabolic phases: START and budding (i.e., early S phase; Figures S4D–S4F) consistently occurred at the ascending part of the oscillating NAD(P)H signal. Mitotic exit always occurred at the signal trough (Figures 4A, 2B, and 2C). ATP oscillations were shifted by ∼180° (Figures S2C and S2D), with budding occurring at the descending part of the ATP signals (Figures S1E–S1H). Next, we examined whether this pattern of phase gating is maintained under other nutrient conditions. We found that at decreased compromise frequencies, START and the early S phase shifted to later NAD(P)H phases (Figures 4B and 4C). In contrast, M exit always occurred at the troughs of the NAD(P)H oscillations, thus exhibiting a strict, condition-independent phase synchrony with metabolism (Figures 4B and 4C). The condition-independent gating of the late cell cycle (M exit) indicates its strong coupling to the metabolic oscillator. Adversely, the frequency-dependent phase gating of the early cell-cycle elements (START/early S) indicates a weaker coupling. Next, to test the robustness of the phase gating, we dynamically perturbed metabolism by switching cells from low (0.01 gL−1) to high (10 gL−1) glucose and recorded their cell-cycle events. Upon the nutrient upshift, all cells responded with a strong, synchronously occurring peak in the NAD(P)H levels (Figure 4D), independently of their stage in the cell cycle (Figure 4E). Thereafter, the NAD(P)H oscillations continued with periods (START to M exit) matching those on high glucose (Figure 4E). Remarkably, those cells that had completed M exit before the nutrient shift (Figure 4E, cells 25–40), consistent with the phase gating under steady-state conditions (Figure 4B), would START only at the ascending part of the NAD(P)H oscillation following the metabolic response (Figure 4F, left), regardless of the timing of their prior M exit (Figure 4E, cells 25–40). Cells interrupted by the nutrient switch after START (Figure 4E, cells 1–16) showed significantly longer (two-tailed t test, p value < 0.001) cell-cycle duration, even when compared to those cultured on low glucose (Figure 4G, gray versus red symbols). Consistent with the observed steady-state and frequency-independent phase gating of M exit (Figure 4C), cells would only exit mitosis at the trough of their NAD(P)H oscillation following the metabolic response (Figure 4F, right), which prolonged their cell cycle (Figure 4G). Our results show that the gating of the cell-cycle phases by the metabolic oscillator is also maintained during dynamic metabolic perturbations and thus is robust. As a result, cell-cycle events are delayed even during nutrient upshifts, waiting for the “right” metabolic phase to occur after the metabolic perturbation, in order to maintain synchrony with the metabolic oscillator. Together, the proportionality among the natural metabolic and compromise frequencies, the critical bandwidth of natural metabolic frequencies required for coupling and cell-cycle initiation, and the phase gating of the cell-cycle phases on the metabolic oscillator indicate that the metabolic oscillator and the cell-cycle oscillator form a system of coupled oscillators. Further, the autonomous nature of the metabolic oscillator, the robust phase gating of the cell-cycle events even during dynamic metabolic perturbations, and the dependency of cell-cycle initiation on the metabolic frequency suggest that the oscillating metabolism is an indispensable component in the cell-cycle regulation machinery, determining the timing of the cell-cycle phases and setting the pace of cell division. On the basis of our data, we derived an interaction topology for the system of coupled oscillators, where the metabolic oscillator is coupled to and gates the phase of the early cell cycle (biomass duplication) and the late cell cycle (biomass segregation) (Figure 5A), operating in addition to the classic C" @default.
• W2562645716 created "2017-01-06" @default.
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• W2562645716 date "2017-01-01" @default.
• W2562645716 modified "2023-10-12" @default.
• W2562645716 title "Autonomous Metabolic Oscillations Robustly Gate the Early and Late Cell Cycle" @default.
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