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Reliable imaging of ATP in living budding and fission yeastMasak Takaine1,2,*, Masaru Ueno3,4, Kenji Kitamura5, Hiromi Imamura6 and Satoshi Yoshida1,2,7,8,*

ABSTRACTAdenosine triphosphate (ATP) is a main metabolite essential for allliving organisms. However, our understanding of ATP dynamicswithin a single living cell is very limited. Here, we optimized theATP-biosensor QUEEN and monitored the dynamics of ATP withgood spatial and temporal resolution in living yeasts. We found stablemaintenance of ATP concentration in wild-type yeasts, regardless ofcarbon sources or cell cycle stages, suggesting that mechanismexists to maintain ATP at a specific concentration. We further foundthat ATP concentration is not necessarily an indicator of metabolicactivity, as there is no clear correlation between ATP level andgrowth rates. During fission yeast meiosis, we found a reductionin ATP levels, suggesting that ATP homeostasis is controlled bydifferentiation. The use of QUEEN in yeasts offers an easy andreliable assay for ATP dynamicity and will answer severalunaddressed questions about cellular metabolism in eukaryotes.

KEY WORDS: ATP, Carbon metabolism, Homeostasis, Yeast,Metabolism, Meiosis, Mitochondria

INTRODUCTIONAdenosine triphosphate (ATP) is a universal energy currency usedby all living organism. In the human body, the half-life of ATP isestimated to be a few seconds (Mortensen et al., 2011), indicatinghigh demand of this energy currency and suggesting that itssynthesis and consumption rates are tightly regulated. Because ofits importance, the molecular mechanism of ATP synthesis byglycolysis and mitochondrial respiration has been rigorouslyinvestigated (Berg et al., 2012; Lehninger et al., 2010). However,little is understood how an ATP concentration is maintained in asingle cell under different conditions because our knowledge onATP dynamics is largely based on biochemical analysis, whichhas poor time resolution compared with the rapid turnover ofATP. Biochemical analysis also precludes characterization ofheterogeneity of ATP concentration within a population or a tissue.The recent development of ATP-biosensors enabled us to monitor

changes inATP concentration in single living cells (reviewed inDongand Zhao, 2016). ATeam, the first FRET-based ATP biosensor, hassuccessfully been introduced intomammalian cells and is nowwidely

used to visualize ATP dynamics in living cells (Imamura et al., 2009).The second generation ATP biosensor QUEEN (Yaginuma et al.,2014) uses a single green fluorescent protein (FP) , instead of thecombination of cyan FP and yellow FP used for ATeam, and hassubstantial advantages, especially for the use in rapidly growingmicroorganisms, such as yeast. First, it has no maturation time lagbetween two FPs that can yield a dysfunctional sensor in rapidlydividing cells, such as bacteria and yeasts (see Discussion for details).Second, it is more resistant to degradation than the FRET-basedsensor ATeam. Third, QUEEN has a 1.7 times wider dynamicrange (the ratio between the maximum and minimum ATPconcentration values) compared with that of ATeam sensors(Yaginuma et al., 2014).

By using QUEEN, it was reported that unexpectedly broadvariations of ATP concentration exist within a clonal population ofbacteria (Yaginuma et al., 2014). In addition to negative-feedbackregulation of a metabolite concentration (Chubukov et al., 2014),eukaryotic cells harbor energy-sensing mechanisms, such asAMP-activated protein kinase (AMPK) (Hardie et al., 2016).Thus, it is expected that the ATP concentration is maintained at aspecific concentration in eukaryotes.

Yeasts have provided an excellent model system for studyingeukaryotic biology. Especially, the central carbon metabolism,including glycolysis and the tricarboxylic acid (TCA) cycle, ofyeast has been extensively studied, and even engineered, becauseof its importance to the fermentation industry to produce usefulmetabolites, including ethanol (Borodina and Nielsen, 2014;Gibson et al., 2017). Yeast carbon metabolism also provides atractable model for the energy metabolism of cancer cells since yeastand cancer cells are similar in that they both mostly synthesize ATPthrough glycolysis, even in the presence of oxygen, as long asglucose supply is high. This is known as ‘aerobic fermentation’ or‘Warburg effect’ in cancer cells (Diaz-Ruiz et al., 2011). In additionto being an important indicator of cell energy, ATP itself is acommon regulator of multiple glycolytic enzymes (Larsson et al.,2000; Mensonides et al., 2013). Therefore, to elucidate thecellular dynamics of ATP is essential also in order to decipher theregulation of glycolytic flux, but remained unaddressed foraforementioned reasons.

Here, we have applied QUEEN in budding and fission yeasts forthe first time, and found that the ATP level showed little variationwithin a population, suggesting a robust ATP homeostasis ineukaryotic cells. We further found that the concentration of ATP ismaintained at a constant level regardless of the carbon sources, andhas no obvious correlation with the mitotic growth phase andgrowth rates. However, we found that ATP levels decline duringfission yeast meiosis, suggesting that ATP homeostasis is controlledwithin a developmental context, not by the availability of sugar.

Taken together, visualization of ATP dynamics in yeast revealsthe existence of robust ATP homeostasis. QUEEN-expressing yeastcells are useful tools to study metabolic activity in individual cells,and offer opportunities to test ATP dynamics under variousenvironmental conditions and in various mutants.Received 5 February 2019; Accepted 4 March 2019

1Gunma University Initiative for Advanced Research (GIAR), Gunma University,Maebashi 371-8512, Japan. 2Institute for Molecular andCellular Regulation (IMCR),Gunma University, Maebashi 371-8512, Japan. 3Department of MolecularBiotechnology, Graduate School of Advanced Sciences of Matter, HiroshimaUniversity, Japan. 4Research Center for the Mathematics on Chromatin LiveDynamics, Hiroshima University, Japan. 5Center for Gene Science, HiroshimaUniversity, 1-4-2 Kagamiyama, Higashi-Hiroshima 739-8527, Japan. 6Departmentof Functional Biology, Graduate School of Biostudies, Kyoto University,Kyoto 606-8501, Japan. 7School of International Liberal Studies, Waseda University,Tokyo, 169-8050, Japan. 8Japan Science and Technology Agency, PREST.

*Authors for correspondence (masaktakaine@gunma-u.ac.jp; satosh@waseda.jp)

M.T., 0000-0002-1279-9505

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© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs230649. doi:10.1242/jcs.230649

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RESULTSQUEEN is a reliable ATP biosensor in budding yeast cellsTo monitor the dynamics of ATP concentration in living single cells,we have recently developed several ATP indicators including ATeam(Imamura et al., 2009) and QUEEN (Yaginuma et al., 2014). Toexplore ATP homeostasis in budding yeast Saccharomyces

cerevisiae, we chose QUEEN because it has several strongpoints(see Introduction). The unique feature of the fluorescent biosensorQUEEN is that the binding to ATP shifts its optimal excitationwavelength from 480 nm to 410 nm (Fig. 1A), which allows us toestimate the ATP level by quantification of the ratio between thefluorescence signal intensities excited at 410 nm and 480 nm.

Fig. 1. See next page for legend.

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We created a budding yeast strain stably expressing QUEEN-2m(Kd of QUEEN-2mwith ATP is∼4.5 mM at 25°C, comparable withestimated cellular ATP concentration in glucose grown yeast) underthe promoter of translation elongation factor 1α (TEF1) from theHIS3 locus. The QUEEN protein was expressed at similar levels in aclonal population and evenly distributed both in the cytoplasm andnucleus due to its small size (42 kDa) but excluded from the vacuole(Fig. 1B). QUEEN has two excitation peaks (at 410 nm and 480 nm;hereafter referred to as 410ex and 480ex, respectively) and oneemission peak (at ∼520 nm). QUEEN was sequentially excited by480 nm and 410 nm light, and the emitted fluorescence signals ataround 520 nm were imaged. The ratio of the emission intensity atthe two excitation peaks (denoted 410ex:480ex) was calculated foreach pixel. The mean of the QUEEN fluorescence intensity ratios(hereafter referred to as the QUEEN ratio) in the intracellular regionreflects the ATP concentration of the cell (see Materials andMethods for details).First, we tested if the QUEEN ratio, indeed, reflects the cellular

concentration of ATP in living yeast cells. A previous biochemicalstudy has demonstrated that ATP levels in budding yeast cellsdropped to 15% upon glucose depletion and gradually recovered to50% of the original level within 20 min (Xu and Bretscher, 2014).Moreover, replacement of glucose in medium with 2-deoxy-D-glucose (2DG), which strongly inhibits glycolysis, reduced cellularATP levels to <1% at least for 30 min (Serrano, 1977; Xu andBretscher, 2014). The QUEEN ratio in yeast cells rapidly droppedafter glucose removal (Fig. 1C, 3 min) but partially recovered within30 min (Fig. 1C). In addition, replacement of glucose with 2% 2DGresulted in rapid and prolonged reduction in the QUEEN ratio(Fig. 1C). Thus, the QUEEN ratio nicely reflects cellular ATPconcentration analyzed by biochemical methods.Second, we tested if QUEEN can reversibly monitor a change in

ATP concentration. We took advantage of gph1Δ mutant cells thatcannot catabolize glycogen and do not show any recovery of ATP

after glucose depletion (Xu and Bretscher, 2014). The meanQUEEN ratios in glucose-depleted gph1Δ cells declined rapidlywithin 10-15 min, but re-feeding of glucose fully recovered thesevalues within a minute (Fig. 1D), indicating that the reduction ofQUEEN ratio after glucose depletion was not due to an irreversibledamage caused to QUEEN. Taken together, these results suggestthat the QUEEN ratio reliably reflects ATP levels in individual cells,allowing us to monitor the dynamicity of ATP concentration inliving cells. Based on the QUEEN ratio, we were also able toestimate the actual ATP concentration in cells by fitting the acquiredQUEEN ratio with the calibration curve (Yaginuma et al., 2014)(Fig. S1 and see Materials and Methods for details).

One of the advantages of using QUEEN compared with classicbiochemical measurements is in its time resolution. We can nowmonitor the dynamic change of ATP concentration in seconds,which allows us to measure metabolic activity of individual livingyeast cells. An example is shown in Fig. 1E, wherewemonitored theQUEEN ratio after the treatment with 2DG. This analysis suggeststhat the half-life of ATP under glycolytic conditions is <1 min inbudding yeast.

We also have generated QUEEN constructs targeting themitochondrial matrix (mitQUEEN) and the cytoplasmic surface ofthe endoplasmic reticulum (ER) (erQUEEN) to analyze the localATP concentration (Fig. 1F). In the case of mitochondria, we foundthat the QUEEN ratio (ATP concentration) in mitochondria is lessthan that in the cytoplasm when cells were grown under glycolyticconditions containing 2% glucose (Fig. S2), which is similar towhathas been reported in HeLa cells by using ATeam (Imamura et al.,2009). The detailed use of mitQUEEN and erQUEEN will bedescribed elsewhere and we focus here on the levels of ATP in thecytoplasm and nucleus. Our results collectively demonstrated thatQUEEN is a useful ATP reporter in budding yeast.

Contribution of carbon sources and respiration toATP concentrationWith the QUEEN system, we are now able to monitor ATP levelsin single living cells under various conditions. First, to examinethe use of carbon sources on ATP, we analyzed QUEEN ratios incells grown in different hexoses. None of the hexoses tested (2%fructose, 2% galactose, 2% glucose, 2% mannose) significantlyaffected ATP concentration (Fig. 2A), suggesting that the ATPlevel is maintained stable, regardless of the carbon sources. Wealso examined the contribution of mitochondrial respiration toATP levels by treatment with the respiration inhibitor antimycin A(Walther et al., 2010). Antimycin A treatment (2 µg/ml) onlyslightly reduced (∼20%) the ATP levels in cells growing in thepresence of one of the hexoses (Fig. 2A), suggesting thatrespiration has a relatively minor role in ATP synthesis if thereis enough carbon.

Next, we explored the effect of fermentative or non-fermentativecarbon sources on ATP levels. Depletion of fermentative carbonsource glucose resulted in a rapid drop of QUEEN ratio within3 min, followed by a gradual recovery after 3 h (Fig. 2B). Thisrecovery was due to activation of respiration because treatment ofantimycin A suppressed the QUEEN ratio (Fig. 2B). When cellswere grown in non-fermentative glycerol, the ATP level wascomparable to that in a glucose-grown culture, and removal of theglycerol had small effect on the QUEEN ratio, even after 3 h. Thehigh ATP level maintained in glycerol grown cells was due to activerespiration, as treatment with antimycin A significantly suppressedit (Fig. 2B). Altogether, our visualization of ATP using QUEENconfirmed that yeast cells growing in fermentative carbon depend

Fig. 1. QUEEN is a reliable ATP biosensor in the budding yeast cells.(A) Design of QUEEN. Signal intensity of ATP-bound QUEEN is highestat 410 nm, whereas that of ATP-free QUEEN is highest at 480 nm.(B) Fluorescence images of yeast cells expressing QUEEN. The greenfluorescence signal was imaged by excitation at 410 nm or 480 nm. TheQUEEN ratio (410 nm ex/480 nm ex) was calculated from the signal intensity ofeach pixel, to generate the QUEEN ratio image of cells. The QUEEN ratio ispseudo-colored to reflect its value throughout the paper. Insets show 2.5-timesmagnified images of the boxed region. (C) Time course analysis of the QUEENratio after glucose depletion. MTY3255 cells grown in SC medium containing2% glucose were washed and released in medium without glucose (top) ormedium containing 2% 2-deoxy-D-glucose (2DG) (bottom). QUEEN signals,excited at 410 nm and 480 nm, and imaged at the indicated time points.Representative images showing QUEEN ratios are shown in the left and themean QUEEN ratios inside the cell were plotted in the right. Horizontal barsindicate averages.N=101–307 cells. (D) QUEEN can report not only reductionbut also recovery of ATP concentration. MTY3153 cells grown in the 2%glucose were released in the medium lacking glucose. After 15 min, 2%glucose was added back to the medium and the QUEEN signal was imaged atthe indicated time points. Left: representative images; right: dot plot of meanQUEEN ratios within the cells. N=137–225 cells. (E) Rapid decrease of theQUEEN ratio in cells treated with glycolytic inhibitor. The QUEEN ratio inMTY3264 cells grown in the 2% glucose was sequentially monitored, followedby supplementation with 2DG just after t=270 s (indicated by the arrow) to givea final concentration of 22 mM glucose and 96 mM 2DG. Representative time-lapse images are shown in the top panel. Plotted QUEEN ratios at each timepoint are shown in the bottom panel. Data are the mean±s.d. (shaded area),collected from three fields of view. (F) Examples of organelle-localizedQUEEN. QUEEN targeted to mitochondria (top) and the ER (bottom). Forcomparison, a cell expressing QUEEN in the cytoplasm was included(indicated by an asterisk). Scale bars: 5 µm.

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heavily on glycolysis to yield cellular ATP (Barnett and Entian,2005). Respiration, however, is highly dominant in yeast cellsgrown in a non-fermentative carbon source (Kayikci and Nielsen,2015; Shashkova et al., 2015) consistent with the observation thatglycerol-grown yeast has highly developed mitochondria comparedto glucose-grown yeast (Egner et al., 2002).

ATP concentration during the cell cycle in budding yeastNext, we analyzed ATP concentration during mitotic cell cycle. TheQUEEN ratio was followed over several generations using time-lapseimaging with Myo1-mCherry as a bud neck marker indicative ofbudded stages. In clear contrast to what has been observed in bacteria(Yaginuma et al., 2014), we observed little fluctuation of ATP levelsin rapidly growing yeast in 2%glucose (Fig. 3A,B;Movie 1).We alsoquantified the QUEEN ratio in cells of different cell cycle stages,such as unbudded (G1), small budded (S) or large budded (G2 andM) but did not find significant differences between cell cycle stages(Fig. 3C). Little fluctuation of ATP concentration during the cellcycle in single cells and among a population suggest a mechanismthat stably maintains the ATP concentration in yeast. Stable

maintenance of ATP during the cell cycle was also observed incells cultured in glycerol (Fig. 3D), suggesting that neither glycolysisnor respiration is significantly regulated during the cell cycle.

QUEEN is a reliable ATP biosensor in fission yeastWe then examined the use of QUEEN in fission yeastSchizosaccharomyces pombe. The QUEEN construct was integratedinto the gene expressing 3-isopropylmalate dehydrogenase (Leu1)under the promoter of the translation elongation and termination factoreIF5A (TIF51). QUEEN protein was evenly distributed both in thecytoplasm and in the nucleus (Fig. 4A). The QUEEN ratio wascalculated in the same manner as described for S. cerevisiae, and wefound a relatively high QUEEN ratio in fission yeast cells grown inEMM medium containing 2% glucose. However, this ratio droppedwithin 5 min after replacing the medium with one containing 20 mM2DG and 10 µg/ml antimycin A (Fig. 4A,B), suggesting that theQUEEN ratio is reflecting the intracellular concentration of ATP infission yeast.We also noticed that the half-life of ATP under glycolyticconditions is ∼1-2 min in fission yeast, as judged by a rapid reductionin QUEEN ratio after 2DG treatment (Fig. 4C).

Fig. 2. Budding yeast ATP homeostasis under different growth conditions. (A) ATP concentration in the cells grown in different types of hexose at 2%.(Mean QUEEN ratio of yeast cells grown in SC medium containing hexose type as indicated as the only carbon source.) QUEEN images were taken aftertreatment with 0.05%DMSO (black/top) or 10 µg/ml antimycin A (blue/bottom) for 30 min. QUEEN ratios were converted into ATP concentration (mM), and plotted(right panel). N=198–555 cells. (B) Respiration is the main source of ATP in the absence of fermentable sugar. MTY3255 cells were grown either in SC +2%glucose (glucose) or in SC +3% glycerol +0.1% glucose (glycerol) to mid-log phase. Cells were then washed three times with SC medium lacking anycarbon sources and released in the same medium (− C). After 3 h, medium was changed to SC lacking carbon but containing 10 µg/ml antimycin A (AM).QUEEN signals were imaged at the indicated time points, and QUEEN ratios of each cell were plotted (right panel). N=126–241 cells. Scale bars: 5 µm.

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Contribution of glycolysis and respiration on fissionyeast ATPIt is known that growth of fission yeast largely relies on glycolysiswhen there are high concentrations of glucose, but relies onrespiration for survival when glucose is limited (Takeda et al.,2015). To examine the relative contribution of glycolysis andrespiration in the ATP level, we monitored the QUEEN ratio offission yeast grown in different glucose concentration. In thepresence of 2% or 0.02% glucose, cells contain about 3 mM ATP.Even after 6 h of glucose depletion, cells are viable and maintained2.3 mM ATP (Fig. 5A,B). The contribution of glucose and

mitochondria to the level of ATP was clearly countercorrelated.Treatment of antimycin A had a minor effect on the ATP level incells grown in 2% glucose but a larger effect in those grown in0.02% glucose (Fig. 5A,B). Respiration was essential for ATPproduction in the absence of glucose. Glucose-depleted cellscompletely lost cellular ATP after the treatment with antimycin Afor 10 min (Fig. 5A,B).

Our analysis of ATP concentration is in good accordance of arecent study by Takeda et al., showing that glucose has a dominantrole in cellular energetics and that respiration plays main role whenglucose concentration is severely limited (Takeda et al., 2015).

Fig. 3. ATP concentration in budding yeast maintains stable during themitotic phase of the cell cycle. (A,B) Long-term imaging (up to 165 min) of QUEENsignals over three generations. Images of cells are shown in (A); the ATP concentrations converted from the QUEEN ratios of the two cells are plottedin (B). Dotted lines show the mean ATP concentration. Cell cycle events, such as bud emergence and cell separation, judged by Myo1-mCherry signals, areindicated by arrows (see also Movie 1). (C) Quantification of QUEEN ratios of cells at different budding stages grown in glucose. MTY3255 cells were grownto mid-log phase in SC +2% glucose, and QUEEN signals were imaged. Cells were classified according to their budding pattern, and the QUEEN ratio foreach cell type was plotted. N=61–125 cells. DIC, differential interference contrast image. (D) Quantification of QUEEN ratios of cells at different budding stagesgrown in glycerol. MTY3255 cells were grown to mid-log phase in SC +3% glycerol +0.1% glucose, and QUEEN signals were imaged. Cells were classifiedaccording to their budding pattern, and the QUEEN ratio for each cell type was plotted. N=27–56 cells. Scale bars: 5 µm.

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Thus, QUEEN is a reliable and useful indicator of ATPconcentration in fission yeast.

ATP concentration during mitotic phase of the cell cyclein fission yeastIn the following, we examined whether ATP concentrationfluctuates during the cell cycle in fission yeast. A unique featureof fission yeast is that the stage of the cell cycle is tightly correlatedwith cell length (Mitchison and Nurse, 1985). We quantified theQUEEN ratio in cells of various lengths but did not find a significantrelationship between QUEEN ratio and cell length, suggesting thatATP concentration does not change during the mitotic phase of thecell cycle (Fig. 6A).Time-lapse imaging of the QUEEN ratio in mitotically growing

cells further confirmed that ATP levels fluctuate little during the cellcycle in fission yeast grown in 2% glucose (Fig. 6B,C andMovie 2).

ATP concentration during fission yeast sporulationWe also examined the QUEEN ratio in sporulating fission yeast.Fission yeast undergoes the meiotic phase of the cell cycle whennitrogen is depleted. We first confirmed that depletion of nitrogen(in the presence of 2% glucose) did not affect cellular ATP levels forthe first 30 min (Fig. 7A,C). After 16 h without nitrogen, a mixedpopulation of fission yeast cells underwent various stages of meiosis(Yamashita et al., 2017). We found a partial decline in the QUEENratio in cells undergoing meiosis (Fig. 7A,B). Treatment withantimycin A (for 30 min) or 2DG (for 10 min) revealed that

glycolysis is the main source of ATP during meiosis (Fig. 7A). Aftersporulation, ATP levels declined further in the remnant cytoplasmbut were maintained at higher levels inside the spores (Fig. 7B,D).The ATP level in the spore also seemed to be due to glycolysis butnot to mitochondria, as treatment with 2DG but not antimycin for2 h had a significant effect (Fig. 7B). These results reveal that theATP level is controlled both temporally and spatially duringmeiosis, and that glycolysis plays pivotal role in the ATP synthesisduring meiosis.

DISCUSSIONIn this study, we visualized ATP dynamics in both budding andfission yeast by using the ATP biosensor QUEEN for the first timein eukaryotic cells. In clear contrast to bacterial cells, there is littlefluctuation/variation in the ATP concentration in yeast populationsgrown under the same culture conditions. Furthermore, we foundthat the ATP concentration in yeast is remarkably constantregardless of the carbon sources or the stage of the cell cyclestage, suggesting a robust ATP homeostasis in eukaryotic cells.

We show that QUEENhas awide dynamic range that is optimal forthe physiological concentration of ATP in yeast cells, and haveestablished a reliable and sensitive assay to measure the ATPconcentration in living yeasts. QUEEN-expressing yeast strains andplasmids are publically available from the Yeast Genetic ResourceCentre Japan (YGRC, http://yeast.nig.ac.jp/yeast/top.xhtml). UsingQUEEN to visualize ATP has many advantages. They include: 1) thereversibility of the QUEEN signal allows to monitor the dynamics of

Fig. 4. QUEEN is a reliable ATP biosensor in the fission yeast. (A) Fluorescence images of the fission yeast expressingQUEEN. KSP3769 cells were grown inEMMmedium containing 2%glucose and imaged before and after exposure to 20 mM2DGand 10 µg/ml antimycin A for 5 min. BF, bright-field image. (B) Dot plotof mean QUEEN ratios of cells shown in (A). N=61 (glucose) and 46 (2DG/AM) cells. (C) Rapid reduction of QUEEN ratios in cells treated with glycolysisinhibitor 2DG. The QUEEN ratio in KSP3769 cells grown in 2% glucosewasmonitored sequentially. Representative time-lapse images are shown every 45 s (leftpanel). Just after 270 s growth medium was supplemented with 2DG (indicated by an arrow) to give a final concentration of 22 mM glucose and 96 mM 2DG.QUEEN ratios at each time point were plotted (right panel); data are the mean±s.d. (shaded area), collected from three fields of view. Scale bars: 5 µm.

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ATP concentration in a single living cell; 2) The time resolution whenusing QUEEN is much higher compared with standard biochemicalassays; 3) Monitoring of the subcellular distribution of ATP ispossible by using organelle-targeting QUEEN; 4) Variations in ATPconcentration can be observed during the life of a single cell or in alineage and among a cell population.There have been several attempts to visualize ATP in living yeasts.

One approach, using an aptamer binding ATP, was successful in real-time visualization of ATP in budding yeast (Özalp et al., 2010).However, the use of the aptamer is limited because its introduction tocells is an invasive and hard-to-control step. The FRET-based ATPbiosensor ATeam is widely used in mammalian systems, but isunsuitable for rapidly proliferating yeasts cells (with a doubling timeof 90–100 min) because the long maturation time of the sensor andthe presence of malfunctioning sensors would lead to unreliableoutcomes as observed when ATeam was used in growing bacteria(Yaginuma et al., 2014). We, thus, believe that QUEEN is the bestATP biosensor for yeasts currently available.By using QUEEN, we found that ATP is maintained at a stable

concentration (3–4 mM) regardless of the growth conditions,suggesting that a mechanism exists to maintain ATP concentrationsat a certain level. We also noticed that the mean ATP concentration in

budding yeast cells grown in the presence of 2% glucose (calculatedusing the QUEEN ratio 3.2±0.4 mM; Fig. 2) shows good agreementwith that of intracellularATP concentrations (estimated by biochemicalanalysis; i.e. 4 mM or 4.6 mM; Gauthier et al., 2008; Ljungdahl andDaignan-Fornier, 2012). This demonstrates reliability of QUEEN andthe validity of our calibration method. By using QUEEN, we havepreviously reported a large variation in cellular ATP levels withinrapidly proliferating bacteria (Yaginuma et al., 2014). In contrast tobacteria, we found little variation in the cellular concentration ofATP inyeast under different growth conditions, suggesting a mechanism thatmaintains ATP concentration in eukaryotic cells.

It has been proposed that concentrations of metabolites aregenerally regulated by negative feedback mechanism (Chubukovet al., 2014). Excessive levels of ATP can inhibit glycolysis byallosterically inactivating phosphofructokinases and pyruvate kinases(Larsson et al., 2000; Reibstein et al., 1986). In eukaryotic cells, adecrease of ATP (and increase of AMP) activates AMPK, which isthought to suppress ATP consumption (Hardie et al., 2016). It is ofgreat interest to define the detailed molecular mechanisms leading tostable maintenance of ATP in yeast.

Because ATP is essential for many cellular functions, ATPconcentration is often used as an indicator of cellular activity. It issurprising to find the cellular concentration of ATP to be similar incells grown with or without glucose because both budding andfission yeasts proliferate significantly slower in the absence ofcarbon sources (Takeda et al., 2015; Tyson et al., 1979). Our studyalso revealed that the ATP concentration is stably maintained duringnormal cell cycle in both types of yeast. It is anticipated that the rateof cellular energy consumption is controlled via the cell cyclebecause the rate of cell growth is affected by the cell cycle (Goranovet al., 2009). Moreover, there are various energy-consuming events,such as DNA replication in S-phase and chromosome segregation inmitosis. Our findings suggest a mechanism that maintains theconcentration of ATP at a certain level, regardless of the speed ofgrowth or stage of the cell cycle. Thus, ATP concentration isunlikely to be a direct indicator of metabolic activity and we need tomonitor the ATP turnover-rate in order to measure cell activity. Theuse of QUEEN offers an easy and reliable assay to measure the ATPturnover-rate compared with standard biochemical assays.

QUEEN-expressing yeast enabled us for the first time to monitorthe ATP level during fission yeast meiosis. ATP levels partiallydecline when cells enter meiosis and are maintained at relatively highlevels only inside the spores. The mechanism by which ATP in thespore is maintained requires further investigation. In addition, theineffectiveness of antimycin A against ATP level in spores does notrule out the possibility that mitochondrial respiration also contributesto maintain ATP in spores because the spore membrane might behardly permeable for the drug. The requirement of glycolysis but notrespiration for ATP synthesis during fission yeast meiosis is somewhatconfusing because fission yeast defective in respiration fails tosporulate (Jambhekar and Amon, 2008). This suggest requirement ofmitochondria for sporulation involves steps other than ATP synthesis,such as lipid and amino acid metabolism. Alternatively, respirationmight play a more-dominant role in meiosis of fission yeast undernatural conditions where carbon sources can also be also limited (andin our sporulation assay, 2% glucose was added).

The mechanism on how the decrease of ATP level in meiotic cellsis also unknown, but our analysis suggests a change in glycolyticactivity not respiration as contributing factor. It has been reported thatexpression levels of metabolic enzymes, including glycolyticenzymes, are largely altered during meiosis in fission yeast(Mata et al., 2002; Yamamoto, 1996), suggesting that metabolic

Fig. 5. Contribution of glycolysis and respiration on ATP levels in fissionyeast. (A) KSP3769 cells were initially grown in EMM medium containing 2%glucose, and then transferred into medium containing 0%, 0.02% or 2%glucose for six hours at 30°C. Cells were imaged before and after exposureto 10 µg/ml antimycin A for 10 min. Representative images are shown.(B) QUEEN ratios of cells were converted into concentration of ATP andplotted. N=50–93 cells. Scale bar: 5 µm.

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activity and ATP homeostasis are remodeled during thisdifferentiation process.Live cell imaging of metabolites is a powerful approach to discover

sporadic events that only happen in a fraction of cells within apopulation, or that fluctuate within a single cell. The fact thatbiosensors can also be targeted to organelles or specific subcellularcompartments, allows the measurement of the concentrationmetabolites at a certain location (Imamura et al., 2009). Furtherstudies are needed to clarify the metabolic control of fission yeastmeiosis but the use and application of QUEEN in yeasts is limitless.We are now able to monitor the dynamics of ATP with good spatialand temporal resolution in living yeasts, and can explore variousunaddressed questions regarding ATP levels, such as levels duringdiauxic shift, starvation, stress response, differentiation and aging.Furthermore, the ease of using the QUEEN system to measure ATPconsumption rates allowed us to analyze metabolic activity in singlecells under various conditions. Therefore, the use of QUEEN in yeastscells may bring our understanding of bioenergetics to another level.

MATERIALS AND METHODSYeast strains and plasmidsBudding and fission yeast strains, and plasmids used in this study are listed inTables S1, S2, and S3, respectively. Strains were constructed by using a PCR-based method (Janke et al., 2004) and genetic crosses. The yeast knockoutstrain collection was originally purchased from GE Healthcare (cat#YSC1053). Some plasmids were originally purchased from EUROSCARF(Oberursel, Germany). Construction of the fission yeast strain expressingQUEEN (KSP3769) is described elsewhere (Ito et al., 2019).

QUEEN-expressing budding yeast strains were constructed as follows.First, QUEEN-2m ORF was isolated by cutting pRSETb-QUEEN-2m(Yaginuma et al., 2014) with BamHI and HindIII, and ligated into BamHI/HindIII sites of pSP-G2 (Partow et al., 2010) to yield MTP3051. Next, aSacI/BamHI fragment of S. cerevisiae TEF1 (translation elongation factor1α) promoter from pYM-N19 (Janke et al., 2004; Mumberg et al., 1995) anda BamHI/PvuII fragment of QUEEN-2m ORF flanked with CYC1terminator from MTP3051 were inserted by trimeric ligation into theSacI-EcoRV sites of pRS303 to yieldMTP3067. For expression of QUEEN-2m from the his3 locus, MTP3067 was linearized by PstI and integrated into

Fig. 6. ATP concentration in fission yeast cellsremains constant during mitotic phase of the cellcycle and cell growth. (A) Approximate relationshipbetween cell cycle stages and cell length in fissionyeast with most septated cells being in S-phase (rightpanel). QUEEN ratios of KSP3769 cells grown in 2%glucose are plotted against cell length (right panel).2DG/AM indicates from cells treated with 20 mM 2DGand 10 µg/ml antimycin A for 10 min. N=20–135 cells.(B) QUEEN ratios of KSP3769 cells grown in EMMmedium containing 2% glucose were imaged every5 min for 12 h. Representative images are shown.Scale bar: 5 µm. (C) QUEEN ratios (dark blue anddark red) and cell lengths (pale blue and pale red) ofthe cells shown in B were plotted over time. See alsoMovie 2.

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the his3Δ1 locus of the wild-type strain MTY3015 to yield MTY3255 and3261. Integrations and the copy numbers of QUEEN were confirmed bydiagnostic PCR and western blotting.

A budding yeast strain expressing a QUEEN construct that localizes to themitochondrial matrix was generated as follows. First, a DNA fragmentencoding a two tandem copies of the mitochondrial targeting signalsequence of human cytochrome c oxidase subunit VIII and the first 20amino acid residues of QUEEN-2m was artificially synthesized with codon

optimization for yeasts (Eurofin Genomics, Ebersberg, Germany). Next, theDNA fragment was inserted into the XbaI-EcoRV sites of MTP3067 to yieldMTP3079. This, in turn, was linearized and integrated into the his3Δ1 locusof MTY3015 to yield MTY3227.

A budding yeast strain expressing a QUEEN construct that localizes to thecytoplasmic surface of endoplasmic reticulum was generated as follows.First, a DNA fragment encoding SEC71TMD (Sato et al., 2003) and itsneighboring sequences (40 amino acids), a five-glycine linker and the first

Fig. 7. ATP concentration is spatially and temporally regulated during meiosis and sporulation. (A) ATP levels in fission yeast cells decrease duringmeiosis. MTY1162 cells were suspended in medium lacking nitrogen (− N) for 30 min or 16 h, and QUEEN ratios were imaged. After 16 h of nitrogen starvation,QUEEN ratios were also imaged in untreated cells (mock) and in cells treated with 10 µg/ml antimycin A for 30 min or with 20 mM 2DG for 10 min.(B) Heterogeneity of ATP concentration in sporulating fission yeast cells. Sporulating MTY1162 cells were suspended in EMM−Nmedium without nitrogen (− N)and imaged. The samples were also treated with 10 µg/ml antimycin A or to 20 mM2DG for 2 h. Asci, indicated by lower contrast of their contours in the bright-field(BF) images, with immature spores (im) or mature spores (m). (C) Quantification of the ATP levels visualized in A. The QUEEN ratios of cells under theindicated conditions were plotted. N=28–118 cells. P values indicating statistical significance are shown. (D) Local concentration of ATP in the spores. Lineprofiles of the QUEEN ratio along the cell length in sporulating cells, as indicated by dashed lines in B. Scale bars: 5 µm.

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20 amino acid sequence of QUEEN-2mwas artificially synthesized (EurofinGenomics). Next, the DNA fragment was inserted into the XbaI-EcoRVsites of MTP3067 to yield MTP3080. MTP3080 was linearized andintegrated into the his3Δ1 locus of MTY3015 to yield MTY3229.

All new strains and plasmids have been deposited to and are availablefrom the Yeast Genetic Resource Centre Japan (YGRC, http://yeast.nig.ac.jp/yeast/top.xhtml).

Media and cell cultureSynthetic completemedium (SC) for budding yeast was prepared according toHanscho et al. (2012). Complete yeast extract (YE) medium, syntheticEdinburgh’s Minimal Medium (EMM) and malt extract (ME) medium forfission yeast were prepared according to the recipes by Forsburg et al.(Forsburg and Rhind, 2006). YE and EMMmedium was supplemented with100 µg/ml adenine, 20 µg/ml uracil, 20 µg/ml L-histidine, 30 µg/ml L-lysineand 60 µg/ml L-leucine. Cells were grown to mid-log phase at 30°C insynthetic medium before imaging unless otherwise indicated. 2-deoxy-D-glucose (2DG) was purchased from FUJIFILMWako (Osaka, Japan) (cat#046-06483) and dissolved in SC or EMM medium instead of glucose.Antimycin A was purchased from FUJIFILM Wako (cat# 514-55521) anddissolved in dimethylsulfoxide (DMSO) tomake a stock solution (20 mg/ml).

To induce mating and meiosis of fission yeast cells, homothallicMTY1162 cells were grown to mid-log in liquid YE, washed twice withwater, and then incubated on an ME plate for 14–20 h at 25°C. Zygotes andasci in the culture were suspended in EMM lacking a nitrogen source(EMM−N), and subjected to microscopy.

MicroscopyBudding and fission yeast cells were immobilized on a 35-mm-glass-bottomdish (#3971-035, 1.5 thickness, IWAKI, Shizuoka, Japan) coated withconcanavalin A (C-7275, Sigma-Aldrich, St Louis) or soybean lectin(L-1395, Sigma-Aldrich), respectively, unless otherwise indicated. The dishwas filled with excess amount of medium (4.5–5 ml) compared with the cellvolume to minimize changes in chemical compositions of the mediumduring observation. In some cases (Figs 1B-D,F and 3C), cells wereconcentrated by centrifugation and sandwiched between a slide and acoverslip (1.5 thickness, Matsunami, Osaka, Japan). In the latter case,imaging was completed within a few minutes after the preparation. Theimmobilized cells were imaged using an inverted fluorescent microscope(Eclipse Ti-E, Nikon, Tokyo, Japan) equipped with Apo TIRF 100× OilDIC N2/NA 1.49 objective lens and an electron multiplying charge-coupleddevice camera (iXon3 DU897E-CS0-#BV80, Andor-Oxford Instruments,Abingdon, UK) at around 25°C. QUEEN fluorescence emitted around520 nm following excitation at 480 nm and 410 nm was assessed from asingle z-plane using a FITC filter set (Ex465-495/DM505/BA515-555,Nikon) and a custom-made filter set (Ex393-425/DM506/BA516-556,Semrock, New York, US), respectively. We sometimes imaged QUEENfluorescence signals by using an FV-1000 confocal laser scanningmicroscope (Olympus, Tokyo, Japan) (Figs 1B-D, 2B and 3C) equippedwith UPLSAPO 100×O/NA 1.4 objective lens (Olympus) with excitation by473-nm and 405-nm lasers using a dichroic mirror DM405/473 and a barrierfilter BA490-540. QUEEN fluorescent signal from mitochondrial matrixwas collected from stacks of eleven z-sections spaced by 0.5 µm. Images ofcells were acquired from several fields of view for each experimentalcondition, providing a high enough sample size for quantitative analysis.

Data analysis and calculation of QUEEN ratioAcquired digital images were analyzed using a Fiji software (Schindelinet al., 2012). The fluorescence images of QUEEN upon excitation at∼480 nm (ex480 image) or ∼410 nm (ex410 image) were collected asdescribed above. The QUEEN ratio was calculated as follows. First, both theQUEEN images were converted to signed 32-bit floating-point grayscaleand then corrected for background using the rolling-ball algorithm or bysubtracting the mean pixel values in an area outside the cells. Next, theimages were thresholded using the modified IsoData algorithm to setbackground (non-thresholded) pixels to the Not a Number (NaN) value. Thepixel values of the ex410 imagewere divided by those of the ex480 image tocalculate the QUEEN ratio at each pixel. The ratio images were expressed in

pseudocolor with appropriate look-up tables and display range. The meanratio in pixels corresponding to the inside of a cell was used to represent theATP level of the cell.

Cell boundaries in the ratio image were determined as follows. QUEENimages were merged, and then thresholded to generate a binary image.Particles (corresponding to cells expressing QUEEN) in the binary imagewere analyzed and automatically outlined to draw regions of interest(ROIs) of the cells. Alternatively, ROIs of cells were manually drawn.Numerical data were plotted using the KaleidaGraph software ver. 4.5.1(Synergy Software, PA, US) or the R studio software ver. 3.4.1 (R CoreTeam, 2017).

Estimation of ATP concentration in yeast from the QUEEN ratiosBased on the biochemical data (Serrano, 1977; Xu and Bretscher, 2014), weassumed depletion of ATP when cells were treated with 2DG in buddingyeast. We quantified the QUEEN ratio in the cells treated with 2DG in theabsence of glucose with our Eclipse Ti-E, Nikon microscope system(Fig. S1A,B) and adjusted the calibration curve previously published(Yaginuma et al., 2014) to the zero-point. This calibration curve was used toestimate the ATP concentration in yeast cells when the QUEEN ratio wasanalyzed with the Nikon system.

The QUEEN ratio in the budding yeast cells in 2% glucose medium was∼2.7 times higher than that in the cells treated with 2DG. Therefore, thedynamic range of the QUEEN ratio is at least 2.7 below our experimentalconditions. This is in good agreementwith previous results, showing a dynamicrange of QUEEN in bacteria of at least threefold (Yaginuma et al., 2014).

Statistical analysisMeans, SDs, and P-values were calculated using Excel software (Microsoft,WA). Significance between two sets of data was tested using unpaired one-tailed Welch’s t-test and is indicated by the low P value (<0.05). Thehorizontal bar in dot plot indicates the average of each population. Weplotted and compared data obtained from experiments carried out the sameday and did not pool data from experiments carried out on different daysbecause several factors vary slightly day-to-day and can affect QUEEN ratio(e.g. room temperature, batch of medium, intensity of excitation light,conditions of optical filters for fluorescence microscopy and cell density).All QUEEN ratio measurements were repeated at least twice.

AcknowledgementsWe thank members of Yoshida/Takaine labs for their support. We also thankK. Ohashi, R. Chaleckis and F. Matsuda for valuable discussion and comments, andthe Yeast Genetic Resource Center (Osaka City University, Japan) for providingplasmids.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: M.T., S.Y.; Methodology: M.T.; Validation: M.T., M.U., K.K.;Formal analysis: M.T.; Investigation: M.T., M.U., S.Y.; Resources: M.T., K.K., H.I.;Data curation: M.T., M.U.; Writing - original draft: M.T., S.Y.; Writing - review &editing: M.T., M.U., K.K., H.I., S.Y.; Supervision: M.T., S.Y.; Project administration:M.T., S.Y.; Funding acquisition: M.T., M.U., S.Y.

FundingThis work was supported by grants from the Japan Society for the Promotion ofScience (JSPS) (grant no. 16H04781 to S.Y. and M.T., and grant no. 15K18525 toM.T.) and Takeda Science foundation (S.Y.). This work was also supported by a jointresearch program of the Institute for Molecular and Cellular Regulation, GunmaUniversity, Japan (M.U. and M.T.).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.230649.supplemental

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