Reliable Imaging of ATP in Living Budding and Fission Yeast Masak Takaine1,2,*, Masaru Ueno3,4, Kenji Kitamura5, Hiromi Imamura6 and Satoshi Yoshida1,2,7,8,*

Home , Aerobic fermentation

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

ABSTRACT used to visualize ATP dynamics in living cells (Imamura et al., 2009). Adenosine triphosphate (ATP) is a main metabolite essential for all The second generation ATP biosensor QUEEN (Yaginuma et al., living organisms. However, our understanding of ATP dynamics 2014) uses a single green fluorescent protein (FP) , instead of the within a single living cell is very limited. Here, we optimized the combination of cyan FP and yellow FP used for ATeam, and has ATP-biosensor QUEEN and monitored the dynamics of ATP with substantial advantages, especially for the use in rapidly growing good spatial and temporal resolution in living yeasts. We found stable microorganisms, such as yeast. First, it has no maturation time lag maintenance of ATP concentration in wild-type yeasts, regardless of between two FPs that can yield a dysfunctional sensor in rapidly carbon sources or cell cycle stages, suggesting that mechanism dividing cells, such as bacteria and yeasts (see Discussion for details). exists to maintain ATP at a specific concentration. We further found Second, it is more resistant to degradation than the FRET-based that ATP concentration is not necessarily an indicator of metabolic sensor ATeam. Third, QUEEN has a 1.7 times wider dynamic activity, as there is no clear correlation between ATP level and range (the ratio between the maximum and minimum ATP growth rates. During fission yeast meiosis, we found a reduction concentration values) compared with that of ATeam sensors in ATP levels, suggesting that ATP homeostasis is controlled by (Yaginuma et al., 2014). differentiation. The use of QUEEN in yeasts offers an easy and By using QUEEN, it was reported that unexpectedly broad reliable assay for ATP dynamicity and will answer several variations of ATP concentration exist within a clonal population of unaddressed questions about cellular metabolism in eukaryotes. bacteria (Yaginuma et al., 2014). In addition to negative-feedback regulation of a metabolite concentration (Chubukov et al., 2014), KEY WORDS: ATP, Carbon metabolism, Homeostasis, Yeast, eukaryotic cells harbor energy-sensing mechanisms, such as Metabolism, Meiosis, Mitochondria AMP-activated protein kinase (AMPK) (Hardie et al., 2016). Thus, it is expected that the ATP concentration is maintained at a INTRODUCTION specific concentration in eukaryotes. Adenosine triphosphate (ATP) is a universal energy currency used Yeasts have provided an excellent model system for studying by all living organism. In the human body, the half-life of ATP is eukaryotic biology. Especially, the central carbon metabolism, estimated to be a few seconds (Mortensen et al., 2011), indicating including glycolysis and the tricarboxylic acid (TCA) cycle, of high demand of this energy currency and suggesting that its yeast has been extensively studied, and even engineered, because synthesis and consumption rates are tightly regulated. Because of of its importance to the fermentation industry to produce useful its importance, the molecular mechanism of ATP synthesis by metabolites, including ethanol (Borodina and Nielsen, 2014; glycolysis and mitochondrial respiration has been rigorously Gibson et al., 2017). Yeast carbon metabolism also provides a investigated (Berg et al., 2012; Lehninger et al., 2010). However, tractable model for the energy metabolism of cancer cells since yeast little is understood how an ATP concentration is maintained in a and cancer cells are similar in that they both mostly synthesize ATP single cell under different conditions because our knowledge on through glycolysis, even in the presence of oxygen, as long as ATP dynamics is largely based on biochemical analysis, which glucose supply is high. This is known as ‘aerobic fermentation’ or has poor time resolution compared with the rapid turnover of ‘Warburg effect’ in cancer cells (Diaz-Ruiz et al., 2011). In addition ATP. Biochemical analysis also precludes characterization of to being an important indicator of cell energy, ATP itself is a heterogeneity of ATP concentration within a population or a tissue. common regulator of multiple glycolytic enzymes (Larsson et al., The recent development of ATP-biosensors enabled us to monitor 2000; Mensonides et al., 2013). Therefore, to elucidate the changes in ATP concentration in single living cells (reviewed in Dong cellular dynamics of ATP is essential also in order to decipher the and Zhao, 2016). ATeam, the first FRET-based ATP biosensor, has regulation of glycolytic flux, but remained unaddressed for successfully been introduced into mammalian cells and is now widely aforementioned reasons. Here, we have applied QUEEN in budding and fission yeasts for the first time, and found that the ATP level showed little variation 1Gunma University Initiative for Advanced Research (GIAR), Gunma University, Maebashi 371-8512, Japan. 2Institute for Molecular and Cellular Regulation (IMCR), within a population, suggesting a robust ATP homeostasis in Gunma University, Maebashi 371-8512, Japan. 3Department of Molecular eukaryotic cells. We further found that the concentration of ATP is Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima maintained at a constant level regardless of the carbon sources, and University, Japan. 4Research Center for the Mathematics on Chromatin Live Dynamics, Hiroshima University, Japan. 5Center for Gene Science, Hiroshima has no obvious correlation with the mitotic growth phase and University, 1-4-2 Kagamiyama, Higashi-Hiroshima 739-8527, Japan. 6Department growth rates. However, we found that ATP levels decline during of Functional Biology, Graduate School of Biostudies, Kyoto University, fission yeast meiosis, suggesting that ATP homeostasis is controlled Kyoto 606-8501, Japan. 7School of International Liberal Studies, Waseda University, Tokyo, 169-8050, Japan. 8Japan Science and Technology Agency, PREST. within a developmental context, not by the availability of sugar. Taken together, visualization of ATP dynamics in yeast reveals *Authors for correspondence ([email protected]; [email protected]) the existence of robust ATP homeostasis. QUEEN-expressing yeast M.T., 0000-0002-1279-9505 cells are useful tools to study metabolic activity in individual cells, and offer opportunities to test ATP dynamics under various

Received 5 February 2019; Accepted 4 March 2019 environmental conditions and in various mutants. Journal of Cell Science

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Fig. 1. See next page for legend.

RESULTS cerevisiae, we chose QUEEN because it has several strongpoints QUEEN is a reliable ATP biosensor in budding yeast cells (see Introduction). The unique feature of the fluorescent biosensor To monitor the dynamics of ATP concentration in living single cells, QUEEN is that the binding to ATP shifts its optimal excitation we have recently developed several ATP indicators including ATeam wavelength from 480 nm to 410 nm (Fig. 1A), which allows us to (Imamura et al., 2009) and QUEEN (Yaginuma et al., 2014). To estimate the ATP level by quantification of the ratio between the explore ATP homeostasis in budding yeast Saccharomyces fluorescence signal intensities excited at 410 nm and 480 nm. Journal of Cell Science

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Fig. 1. QUEEN is a reliable ATP biosensor in the budding yeast cells. after glucose depletion (Xu and Bretscher, 2014). The mean (A) Design of QUEEN. Signal intensity of ATP-bound QUEEN is highest QUEEN ratios in glucose-depleted gph1Δ cells declined rapidly at 410 nm, whereas that of ATP-free QUEEN is highest at 480 nm. within 10-15 min, but re-feeding of glucose fully recovered these (B) Fluorescence images of yeast cells expressing QUEEN. The green fluorescence signal was imaged by excitation at 410 nm or 480 nm. The values within a minute (Fig. 1D), indicating that the reduction of QUEEN ratio (410 nm ex/480 nm ex) was calculated from the signal intensity of QUEEN ratio after glucose depletion was not due to an irreversible each pixel, to generate the QUEEN ratio image of cells. The QUEEN ratio is damage caused to QUEEN. Taken together, these results suggest pseudo-colored to reflect its value throughout the paper. Insets show 2.5-times that the QUEEN ratio reliably reflects ATP levels in individual cells, magnified images of the boxed region. (C) Time course analysis of the QUEEN allowing us to monitor the dynamicity of ATP concentration in ratio after glucose depletion. MTY3255 cells grown in SC medium containing living cells. Based on the QUEEN ratio, we were also able to 2% glucose were washed and released in medium without glucose (top) or medium containing 2% 2-deoxy-D-glucose (2DG) (bottom). QUEEN signals, estimate the actual ATP concentration in cells by fitting the acquired excited at 410 nm and 480 nm, and imaged at the indicated time points. QUEEN ratio with the calibration curve (Yaginuma et al., 2014) Representative images showing QUEEN ratios are shown in the left and the (Fig. S1 and see Materials and Methods for details). mean QUEEN ratios inside the cell were plotted in the right. Horizontal bars One of the advantages of using QUEEN compared with classic indicate averages. N=101–307 cells. (D) QUEEN can report not only reduction biochemical measurements is in its time resolution. We can now but also recovery of ATP concentration. MTY3153 cells grown in the 2% monitor the dynamic change of ATP concentration in seconds, glucose were released in the medium lacking glucose. After 15 min, 2% which allows us to measure metabolic activity of individual living glucose was added back to the medium and the QUEEN signal was imaged at the indicated time points. Left: representative images; right: dot plot of mean yeast cells. An example is shown in Fig. 1E, where we monitored the QUEEN ratios within the cells. N=137–225 cells. (E) Rapid decrease of the QUEEN ratio after the treatment with 2DG. This analysis suggests QUEEN ratio in cells treated with glycolytic inhibitor. The QUEEN ratio in that the half-life of ATP under glycolytic conditions is <1 min in MTY3264 cells grown in the 2% glucose was sequentially monitored, followed budding yeast. by supplementation with 2DG just after t=270 s (indicated by the arrow) to give We also have generated QUEEN constructs targeting the a final concentration of 22 mM glucose and 96 mM 2DG. Representative time- mitochondrial matrix (mitQUEEN) and the cytoplasmic surface of lapse images are shown in the top panel. Plotted QUEEN ratios at each time point are shown in the bottom panel. Data are the mean±s.d. (shaded area), the endoplasmic reticulum (ER) (erQUEEN) to analyze the local collected from three fields of view. (F) Examples of organelle-localized ATP concentration (Fig. 1F). In the case of mitochondria, we found QUEEN. QUEEN targeted to mitochondria (top) and the ER (bottom). For that the QUEEN ratio (ATP concentration) in mitochondria is less comparison, a cell expressing QUEEN in the cytoplasm was included than that in the cytoplasm when cells were grown under glycolytic (indicated by an asterisk). Scale bars: 5 µm. conditions containing 2% glucose (Fig. S2), which is similar to what has been reported in HeLa cells by using ATeam (Imamura et al., We created a budding yeast strain stably expressing QUEEN-2m 2009). The detailed use of mitQUEEN and erQUEEN will be (Kd of QUEEN-2m with ATP is ∼4.5 mM at 25°C, comparable with described elsewhere and we focus here on the levels of ATP in the estimated cellular ATP concentration in glucose grown yeast) under cytoplasm and nucleus. Our results collectively demonstrated that the promoter of translation elongation factor 1α (TEF1) from the QUEEN is a useful ATP reporter in budding yeast. HIS3 locus. The QUEEN protein was expressed at similar levels in a clonal population and evenly distributed both in the cytoplasm and Contribution of carbon sources and respiration to nucleus due to its small size (42 kDa) but excluded from the vacuole ATP concentration (Fig. 1B). QUEEN has two excitation peaks (at 410 nm and 480 nm; With the QUEEN system, we are now able to monitor ATP levels hereafter referred to as 410ex and 480ex, respectively) and one in single living cells under various conditions. First, to examine emission peak (at ∼520 nm). QUEEN was sequentially excited by the use of carbon sources on ATP, we analyzed QUEEN ratios in 480 nm and 410 nm light, and the emitted fluorescence signals at cells grown in different hexoses. None of the hexoses tested (2% around 520 nm were imaged. The ratio of the emission intensity at fructose, 2% galactose, 2% glucose, 2% mannose) significantly the two excitation peaks (denoted 410ex:480ex) was calculated for affected ATP concentration (Fig. 2A), suggesting that the ATP each pixel. The mean of the QUEEN fluorescence intensity ratios level is maintained stable, regardless of the carbon sources. We (hereafter referred to as the QUEEN ratio) in the intracellular region also examined the contribution of mitochondrial respiration to reflects the ATP concentration of the cell (see Materials and ATP levels by treatment with the respiration inhibitor antimycin A Methods for details). (Walther et al., 2010). Antimycin A treatment (2 µg/ml) only First, we tested if the QUEEN ratio, indeed, reflects the cellular slightly reduced (∼20%) the ATP levels in cells growing in the concentration of ATP in living yeast cells. A previous biochemical presence of one of the hexoses (Fig. 2A), suggesting that study has demonstrated that ATP levels in budding yeast cells respiration has a relatively minor role in ATP synthesis if there dropped to 15% upon glucose depletion and gradually recovered to is enough carbon. 50% of the original level within 20 min (Xu and Bretscher, 2014). Next, we explored the effect of fermentative or non-fermentative Moreover, replacement of glucose in medium with 2-deoxy-D- carbon sources on ATP levels. Depletion of fermentative carbon glucose (2DG), which strongly inhibits glycolysis, reduced cellular source glucose resulted in a rapid drop of QUEEN ratio within ATP levels to <1% at least for 30 min (Serrano, 1977; Xu and 3 min, followed by a gradual recovery after 3 h (Fig. 2B). This Bretscher, 2014). The QUEEN ratio in yeast cells rapidly dropped recovery was due to activation of respiration because treatment of after glucose removal (Fig. 1C, 3 min) but partially recovered within antimycin A suppressed the QUEEN ratio (Fig. 2B). When cells 30 min (Fig. 1C). In addition, replacement of glucose with 2% 2DG were grown in non-fermentative glycerol, the ATP level was resulted in rapid and prolonged reduction in the QUEEN ratio comparable to that in a glucose-grown culture, and removal of the (Fig. 1C). Thus, the QUEEN ratio nicely reflects cellular ATP glycerol had small effect on the QUEEN ratio, even after 3 h. The concentration analyzed by biochemical methods. high ATP level maintained in glycerol grown cells was due to active Second, we tested if QUEEN can reversibly monitor a change in respiration, as treatment with antimycin A significantly suppressed ATP concentration. We took advantage of gph1Δ mutant cells that it (Fig. 2B). Altogether, our visualization of ATP using QUEEN cannot catabolize glycogen and do not show any recovery of ATP confirmed that yeast cells growing in fermentative carbon depend Journal of Cell Science

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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 after treatment 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 any carbon 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. heavily on glycolysis to yield cellular ATP (Barnett and Entian, maintenance of ATP during the cell cycle was also observed in 2005). Respiration, however, is highly dominant in yeast cells cells cultured in glycerol (Fig. 3D), suggesting that neither glycolysis grown in a non-fermentative carbon source (Kayikci and Nielsen, nor respiration is significantly regulated during the cell cycle. 2015; Shashkova et al., 2015) consistent with the observation that glycerol-grown yeast has highly developed mitochondria compared QUEEN is a reliable ATP biosensor in fission yeast to glucose-grown yeast (Egner et al., 2002). We then examined the use of QUEEN in fission yeast Schizosaccharomyces pombe. The QUEEN construct was integrated ATP concentration during the cell cycle in budding yeast into the gene expressing 3-isopropylmalate dehydrogenase (Leu1) Next, we analyzed ATP concentration during mitotic cell cycle. The under the promoter of the translation elongation and termination factor QUEEN ratio was followed over several generations using time-lapse eIF5A (TIF51). QUEEN protein was evenly distributed both in the imaging with Myo1-mCherry as a bud neck marker indicative of cytoplasm and in the nucleus (Fig. 4A). The QUEEN ratio was budded stages. In clear contrast to what has been observed in bacteria calculated in the same manner as described for S. cerevisiae,andwe (Yaginuma et al., 2014), we observed little fluctuation of ATP levels found a relatively high QUEEN ratio in fission yeast cells grown in in rapidly growing yeast in 2% glucose (Fig. 3A,B; Movie 1). We also EMM medium containing 2% glucose. However, this ratio dropped quantified the QUEEN ratio in cells of different cell cycle stages, within 5 min after replacing the medium with one containing 20 mM such as unbudded (G1), small budded (S) or large budded (G2 and 2DG and 10 µg/ml antimycin A (Fig. 4A,B), suggesting that the M) but did not find significant differences between cell cycle stages QUEEN ratio is reflecting the intracellular concentration of ATP in (Fig. 3C). Little fluctuation of ATP concentration during the cell fission yeast. We also noticed that the half-life of ATP under glycolytic cycle in single cells and among a population suggest a mechanism conditions is ∼1-2 min in fission yeast, as judged by a rapid reduction that stably maintains the ATP concentration in yeast. Stable in QUEEN ratio after 2DG treatment (Fig. 4C). Journal of Cell Science

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

Contribution of glycolysis and respiration on fission mitochondria to the level of ATP was clearly countercorrelated. yeast ATP Treatment of antimycin A had a minor effect on the ATP level in It is known that growth of fission yeast largely relies on glycolysis cells grown in 2% glucose but a larger effect in those grown in when there are high concentrations of glucose, but relies on 0.02% glucose (Fig. 5A,B). Respiration was essential for ATP respiration for survival when glucose is limited (Takeda et al., production in the absence of glucose. Glucose-depleted cells 2015). To examine the relative contribution of glycolysis and completely lost cellular ATP after the treatment with antimycin A respiration in the ATP level, we monitored the QUEEN ratio of for 10 min (Fig. 5A,B). fission yeast grown in different glucose concentration. In the Our analysis of ATP concentration is in good accordance of a presence of 2% or 0.02% glucose, cells contain about 3 mM ATP. recent study by Takeda et al., showing that glucose has a dominant Even after 6 h of glucose depletion, cells are viable and maintained role in cellular energetics and that respiration plays main role when

2.3 mM ATP (Fig. 5A,B). The contribution of glucose and glucose concentration is severely limited (Takeda et al., 2015). Journal of Cell Science

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Fig. 4. QUEEN is a reliable ATP biosensor in the fission yeast. (A) Fluorescence images of the fission yeast expressing QUEEN. KSP3769 cells were grown in EMM medium containing 2% glucose and imaged before and after exposure to 20 mM 2DG and 10 µg/ml antimycin A for 5 min. BF, bright-field image. (B) Dot plot of 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 glycolysis inhibitor 2DG. The QUEEN ratio in KSP3769 cells grown in 2% glucose was monitored sequentially. Representative time-lapse images are shown every 45 s (left panel). 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.

Thus, QUEEN is a reliable and useful indicator of ATP glycolysis is the main source of ATP during meiosis (Fig. 7A). After concentration in fission yeast. sporulation, ATP levels declined further in the remnant cytoplasm but were maintained at higher levels inside the spores (Fig. 7B,D). ATP concentration during mitotic phase of the cell cycle The ATP level in the spore also seemed to be due to glycolysis but in fission yeast not to mitochondria, as treatment with 2DG but not antimycin for In the following, we examined whether ATP concentration 2 h had a significant effect (Fig. 7B). These results reveal that the fluctuates during the cell cycle in fission yeast. A unique feature ATP level is controlled both temporally and spatially during of fission yeast is that the stage of the cell cycle is tightly correlated meiosis, and that glycolysis plays pivotal role in the ATP synthesis with cell length (Mitchison and Nurse, 1985). We quantified the during meiosis. QUEEN ratio in cells of various lengths but did not find a significant relationship between QUEEN ratio and cell length, suggesting that DISCUSSION ATP concentration does not change during the mitotic phase of the In this study, we visualized ATP dynamics in both budding and cell cycle (Fig. 6A). fission yeast by using the ATP biosensor QUEEN for the first time Time-lapse imaging of the QUEEN ratio in mitotically growing in eukaryotic cells. In clear contrast to bacterial cells, there is little cells further confirmed that ATP levels fluctuate little during the cell fluctuation/variation in the ATP concentration in yeast populations cycle in fission yeast grown in 2% glucose (Fig. 6B,C and Movie 2). grown under the same culture conditions. Furthermore, we found that the ATP concentration in yeast is remarkably constant ATP concentration during fission yeast sporulation regardless of the carbon sources or the stage of the cell cycle We also examined the QUEEN ratio in sporulating fission yeast. stage, suggesting a robust ATP homeostasis in eukaryotic cells. Fission yeast undergoes the meiotic phase of the cell cycle when We show that QUEEN has a wide dynamic range that is optimal for nitrogen is depleted. We first confirmed that depletion of nitrogen the physiological concentration of ATP in yeast cells, and have (in the presence of 2% glucose) did not affect cellular ATP levels for established a reliable and sensitive assay to measure the ATP the first 30 min (Fig. 7A,C). After 16 h without nitrogen, a mixed concentration in living yeasts. QUEEN-expressing yeast strains and population of fission yeast cells underwent various stages of meiosis plasmids are publically available from the Yeast Genetic Resource (Yamashita et al., 2017). We found a partial decline in the QUEEN Centre Japan (YGRC, http://yeast.nig.ac.jp/yeast/top.xhtml). Using ratio in cells undergoing meiosis (Fig. 7A,B). Treatment with QUEEN to visualize ATP has many advantages. They include: 1) the antimycin A (for 30 min) or 2DG (for 10 min) revealed that reversibility of the QUEEN signal allows to monitor the dynamics of Journal of Cell Science

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budding yeast cells grown in the presence of 2% glucose (calculated using the QUEEN ratio 3.2±0.4 mM; Fig. 2) shows good agreement with that of intracellular ATP concentrations (estimated by biochemical analysis; i.e. 4 mM or 4.6 mM; Gauthier et al., 2008; Ljungdahl and Daignan-Fornier, 2012). This demonstrates reliability of QUEEN and the validity of our calibration method. By using QUEEN, we have previously reported a large variation in cellular ATP levels within rapidly proliferating bacteria (Yaginuma et al., 2014). In contrast to bacteria, we found little variation in the cellular concentration of ATP in yeast under different growth conditions, suggesting a mechanism that maintains ATP concentration in eukaryotic cells. It has been proposed that concentrations of metabolites are generally regulated by negative feedback mechanism (Chubukov et al., 2014). Excessive levels of ATP can inhibit glycolysis by allosterically inactivating phosphofructokinases and pyruvate kinases (Larsson et al., 2000; Reibstein et al., 1986). In eukaryotic cells, a decrease of ATP (and increase of AMP) activates AMPK, which is thought to suppress ATP consumption (Hardie et al., 2016). It is of great interest to define the detailed molecular mechanisms leading to stable maintenance of ATP in yeast. Because ATP is essential for many cellular functions, ATP concentration is often used as an indicator of cellular activity. It is surprising to find the cellular concentration of ATP to be similar in cells grown with or without glucose because both budding and fission yeasts proliferate significantly slower in the absence of carbon sources (Takeda et al., 2015; Tyson et al., 1979). Our study also revealed that the ATP concentration is stably maintained during normal cell cycle in both types of yeast. It is anticipated that the rate of cellular energy consumption is controlled via the cell cycle because the rate of cell growth is affected by the cell cycle (Goranov et al., 2009). Moreover, there are various energy-consuming events, such as DNA replication in S-phase and chromosome segregation in Fig. 5. Contribution of glycolysis and respiration on ATP levels in fission mitosis. Our findings suggest a mechanism that maintains the yeast. (A) KSP3769 cells were initially grown in EMM medium containing 2% concentration of ATP at a certain level, regardless of the speed of glucose, and then transferred into medium containing 0%, 0.02% or 2% growth or stage of the cell cycle. Thus, ATP concentration is glucose for six hours at 30°C. Cells were imaged before and after exposure unlikely to be a direct indicator of metabolic activity and we need to to 10 µg/ml antimycin A for 10 min. Representative images are shown. monitor the ATP turnover-rate in order to measure cell activity. The (B) QUEEN ratios of cells were converted into concentration of ATP and plotted. N=50–93 cells. Scale bar: 5 µm. use of QUEEN offers an easy and reliable assay to measure the ATP turnover-rate compared with standard biochemical assays. QUEEN-expressing yeast enabled us for the first time to monitor ATP concentration in a single living cell; 2) The time resolution when the ATP level during fission yeast meiosis. ATP levels partially using QUEEN is much higher compared with standard biochemical decline when cells enter meiosis and are maintained at relatively high assays; 3) Monitoring of the subcellular distribution of ATP is levels only inside the spores. The mechanism by which ATP in the possible by using organelle-targeting QUEEN; 4) Variations in ATP spore is maintained requires further investigation. In addition, the concentration can be observed during the life of a single cell or in a ineffectiveness of antimycin A against ATP level in spores does not lineage and among a cell population. rule out the possibility that mitochondrial respiration also contributes There have been several attempts to visualize ATP in living yeasts. to maintain ATP in spores because the spore membrane might be One approach, using an aptamer binding ATP, was successful in real- hardly permeable for the drug. The requirement of glycolysis but not time visualization of ATP in budding yeast (Özalp et al., 2010). respiration for ATP synthesis during fission yeast meiosis is somewhat However, the use of the aptamer is limited because its introduction to confusing because fission yeast defective in respiration fails to cells is an invasive and hard-to-control step. The FRET-based ATP sporulate (Jambhekar and Amon, 2008). This suggest requirement of biosensor ATeam is widely used in mammalian systems, but is mitochondria for sporulation involves steps other than ATP synthesis, unsuitable for rapidly proliferating yeasts cells (with a doubling time such as lipid and amino acid metabolism. Alternatively, respiration of 90–100 min) because the long maturation time of the sensor and might play a more-dominant role in meiosis of fission yeast under the presence of malfunctioning sensors would lead to unreliable natural conditions where carbon sources can also be also limited (and outcomes as observed when ATeam was used in growing bacteria in our sporulation assay, 2% glucose was added). (Yaginuma et al., 2014). We, thus, believe that QUEEN is the best The mechanism on how the decrease of ATP level in meiotic cells ATP biosensor for yeasts currently available. is also unknown, but our analysis suggests a change in glycolytic By using QUEEN, we found that ATP is maintained at a stable activity not respiration as contributing factor. It has been reported that concentration (3–4 mM) regardless of the growth conditions, expression levels of metabolic enzymes, including glycolytic suggesting that a mechanism exists to maintain ATP concentrations enzymes, are largely altered during meiosis in fission yeast at a certain level. We also noticed that the mean ATP concentration in (Mata et al., 2002; Yamamoto, 1996), suggesting that metabolic Journal of Cell Science

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Fig. 6. ATP concentration in fission yeast cells remains constant during mitotic phase of the cell cycle and cell growth. (A) Approximate relationship between cell cycle stages and cell length in fission yeast with most septated cells being in S-phase (right panel). 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 2DG and 10 µg/ml antimycin A for 10 min. N=20–135 cells. (B) QUEEN ratios of KSP3769 cells grown in EMM medium containing 2% glucose were imaged every 5 min for 12 h. Representative images are shown. Scale bar: 5 µm. (C) QUEEN ratios (dark blue and dark red) and cell lengths (pale blue and pale red) of the cells shown in B were plotted over time. See also Movie 2.

activity and ATP homeostasis are remodeled during this MATERIALS AND METHODS differentiation process. Yeast strains and plasmids Live cell imaging of metabolites is a powerful approach to discover Budding and fission yeast strains, and plasmids used in this study are listed in sporadic events that only happen in a fraction of cells within a Tables S1, S2, and S3, respectively. Strains were constructed by using a PCR- population, or that fluctuate within a single cell. The fact that based method (Janke et al., 2004) and genetic crosses. The yeast knockout biosensors can also be targeted to organelles or specific subcellular strain collection was originally purchased from GE Healthcare (cat# compartments, allows the measurement of the concentration YSC1053). Some plasmids were originally purchased from EUROSCARF metabolites at a certain location (Imamura et al., 2009). Further (Oberursel, Germany). Construction of the fission yeast strain expressing studies are needed to clarify the metabolic control of fission yeast QUEEN (KSP3769) is described elsewhere (Ito et al., 2019). meiosis but the use and application of QUEEN in yeasts is limitless. QUEEN-expressing budding yeast strains were constructed as follows. We are now able to monitor the dynamics of ATP with good spatial First, QUEEN-2m ORF was isolated by cutting pRSETb-QUEEN-2m and temporal resolution in living yeasts, and can explore various (Yaginuma et al., 2014) with BamHI and HindIII, and ligated into BamHI/ unaddressed questions regarding ATP levels, such as levels during HindIII sites of pSP-G2 (Partow et al., 2010) to yield MTP3051. Next, a SacI/BamHI fragment of S. cerevisiae TEF1 (translation elongation factor diauxic shift, starvation, stress response, differentiation and aging. 1α) promoter from pYM-N19 (Janke et al., 2004; Mumberg et al., 1995) and Furthermore, the ease of using the QUEEN system to measure ATP a BamHI/PvuII fragment of QUEEN-2m ORF flanked with CYC1 consumption rates allowed us to analyze metabolic activity in single terminator from MTP3051 were inserted by trimeric ligation into the cells under various conditions. Therefore, the use of QUEEN in yeasts SacI-EcoRV sites of pRS303 to yield MTP3067. For expression of QUEEN- cells may bring our understanding of bioenergetics to another level. 2m from the his3 locus, MTP3067 was linearized by PstI and integrated into Journal of Cell Science

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Fig. 7. ATP concentration is spatially and temporally regulated during meiosis and sporulation. (A) ATP levels in fission yeast cells decrease during meiosis. 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−N medium without nitrogen (− N) and imaged. The samples were also treated with 10 µg/ml antimycin A or to 20 mM 2DG 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 the indicated conditions were plotted. N=28–118 cells. P values indicating statistical significance are shown. (D) Local concentration of ATP in the spores. Line profiles of the QUEEN ratio along the cell length in sporulating cells, as indicated by dashed lines in B. Scale bars: 5 µm. the his3Δ1 locus of the wild-type strain MTY3015 to yield MTY3255 and optimization for yeasts (Eurofin Genomics, Ebersberg, Germany). Next, the 3261. Integrations and the copy numbers of QUEEN were confirmed by DNA fragment was inserted into the XbaI-EcoRV sites of MTP3067 to yield diagnostic PCR and western blotting. MTP3079. This, in turn, was linearized and integrated into the his3Δ1 locus A budding yeast strain expressing a QUEEN construct that localizes to the of MTY3015 to yield MTY3227. mitochondrial matrix was generated as follows. First, a DNA fragment A budding yeast strain expressing a QUEEN construct that localizes to the encoding a two tandem copies of the mitochondrial targeting signal cytoplasmic surface of endoplasmic reticulum was generated as follows. sequence of human cytochrome c oxidase subunit VIII and the first 20 First, a DNA fragment encoding SEC71TMD (Sato et al., 2003) and its amino acid residues of QUEEN-2m was artificially synthesized with codon neighboring sequences (40 amino acids), a five-glycine linker and the first Journal of Cell Science

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20 amino acid sequence of QUEEN-2m was artificially synthesized (Eurofin pseudocolor with appropriate look-up tables and display range. The mean Genomics). Next, the DNA fragment was inserted into the XbaI-EcoRV ratio in pixels corresponding to the inside of a cell was used to represent the sites of MTP3067 to yield MTP3080. MTP3080 was linearized and ATP level of the cell. integrated into the his3Δ1 locus of MTY3015 to yield MTY3229. Cell boundaries in the ratio image were determined as follows. QUEEN All new strains and plasmids have been deposited to and are available images were merged, and then thresholded to generate a binary image. from the Yeast Genetic Resource Centre Japan (YGRC, http://yeast.nig.ac. Particles (corresponding to cells expressing QUEEN) in the binary image jp/yeast/top.xhtml). were analyzed and automatically outlined to draw regions of interest (ROIs) of the cells. Alternatively, ROIs of cells were manually drawn. Media and cell culture Numerical data were plotted using the KaleidaGraph software ver. 4.5.1 Synthetic complete medium (SC) for budding yeast was prepared according to (Synergy Software, PA, US) or the R studio software ver. 3.4.1 (R Core Hanscho et al. (2012). Complete yeast extract (YE) medium, synthetic Team, 2017). Edinburgh’s Minimal Medium (EMM) and malt extract (ME) medium for fission yeast were prepared according to the recipes by Forsburg et al. Estimation of ATP concentration in yeast from the QUEEN ratios (Forsburg and Rhind, 2006). YE and EMM medium was supplemented with Based on the biochemical data (Serrano, 1977; Xu and Bretscher, 2014), we 100 µg/ml adenine, 20 µg/ml uracil, 20 µg/ml L-histidine, 30 µg/ml L-lysine assumed depletion of ATP when cells were treated with 2DG in budding and 60 µg/ml L-leucine. Cells were grown to mid-log phase at 30°C in yeast. We quantified the QUEEN ratio in the cells treated with 2DG in the synthetic medium before imaging unless otherwise indicated. 2-deoxy- absence of glucose with our Eclipse Ti-E, Nikon microscope system D-glucose (2DG) was purchased from FUJIFILM Wako (Osaka, Japan) (cat# (Fig. S1A,B) and adjusted the calibration curve previously published 046-06483) and dissolved in SC or EMM medium instead of glucose. (Yaginuma et al., 2014) to the zero-point. This calibration curve was used to Antimycin A was purchased from FUJIFILM Wako (cat# 514-55521) and estimate the ATP concentration in yeast cells when the QUEEN ratio was dissolved in dimethylsulfoxide (DMSO) to make a stock solution (20 mg/ml). analyzed with the Nikon system. To induce mating and meiosis of fission yeast cells, homothallic The QUEEN ratio in the budding yeast cells in 2% glucose medium was MTY1162 cells were grown to mid-log in liquid YE, washed twice with ∼2.7 times higher than that in the cells treated with 2DG. Therefore, the water, and then incubated on an ME plate for 14–20 h at 25°C. Zygotes and dynamic range of the QUEEN ratio is at least 2.7 below our experimental asci in the culture were suspended in EMM lacking a nitrogen source conditions. This is in good agreement with previous results, showing a dynamic (EMM−N), and subjected to microscopy. range of QUEEN in bacteria of at least threefold (Yaginuma et al., 2014).

Microscopy Statistical analysis Budding and fission yeast cells were immobilized on a 35-mm-glass-bottom Means, SDs, and P-values were calculated using Excel software (Microsoft, dish (#3971-035, 1.5 thickness, IWAKI, Shizuoka, Japan) coated with WA). Significance between two sets of data was tested using unpaired one- concanavalin A (C-7275, Sigma-Aldrich, St Louis) or soybean lectin tailed Welch’s t-test and is indicated by the low P value (<0.05). The (L-1395, Sigma-Aldrich), respectively, unless otherwise indicated. The dish horizontal bar in dot plot indicates the average of each population. We was filled with excess amount of medium (4.5–5 ml) compared with the cell plotted and compared data obtained from experiments carried out the same volume to minimize changes in chemical compositions of the medium day and did not pool data from experiments carried out on different days during observation. In some cases (Figs 1B-D,F and 3C), cells were because several factors vary slightly day-to-day and can affect QUEEN ratio concentrated by centrifugation and sandwiched between a slide and a (e.g. room temperature, batch of medium, intensity of excitation light, coverslip (1.5 thickness, Matsunami, Osaka, Japan). In the latter case, conditions of optical filters for fluorescence microscopy and cell density). imaging was completed within a few minutes after the preparation. The All QUEEN ratio measurements were repeated at least twice. immobilized cells were imaged using an inverted fluorescent microscope (Eclipse Ti-E, Nikon, Tokyo, Japan) equipped with Apo TIRF 100× Oil Acknowledgements DIC N2/NA 1.49 objective lens and an electron multiplying charge-coupled We thank members of Yoshida/Takaine labs for their support. We also thank device camera (iXon3 DU897E-CS0-#BV80, Andor-Oxford Instruments, K. Ohashi, R. Chaleckis and F. Matsuda for valuable discussion and comments, and Abingdon, UK) at around 25°C. QUEEN fluorescence emitted around the Yeast Genetic Resource Center (Osaka City University, Japan) for providing plasmids. 520 nm following excitation at 480 nm and 410 nm was assessed from a single z-plane using a FITC filter set (Ex465-495/DM505/BA515-555, Competing interests Nikon) and a custom-made filter set (Ex393-425/DM506/BA516-556, The authors declare no competing or financial interests. Semrock, New York, US), respectively. We sometimes imaged QUEEN fluorescence signals by using an FV-1000 confocal laser scanning Author contributions microscope (Olympus, Tokyo, Japan) (Figs 1B-D, 2B and 3C) equipped Conceptualization: M.T., S.Y.; Methodology: M.T.; Validation: M.T., M.U., K.K.; with UPLSAPO 100×O/NA 1.4 objective lens (Olympus) with excitation by Formal analysis: M.T.; Investigation: M.T., M.U., S.Y.; Resources: M.T., K.K., H.I.; 473-nm and 405-nm lasers using a dichroic mirror DM405/473 and a barrier Data curation: M.T., M.U.; Writing - original draft: M.T., S.Y.; Writing - review & filter BA490-540. QUEEN fluorescent signal from mitochondrial matrix 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. was collected from stacks of eleven z-sections spaced by 0.5 µm. Images of cells were acquired from several fields of view for each experimental Funding condition, providing a high enough sample size for quantitative analysis. This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) (grant no. 16H04781 to S.Y. and M.T., and grant no. 15K18525 to Data analysis and calculation of QUEEN ratio M.T.) and Takeda Science foundation (S.Y.). This work was also supported by a joint Acquired digital images were analyzed using a Fiji software (Schindelin research program of the Institute for Molecular and Cellular Regulation, Gunma et al., 2012). The fluorescence images of QUEEN upon excitation at University, Japan (M.U. and M.T.). ∼480 nm (ex480 image) or ∼410 nm (ex410 image) were collected as described above. The QUEEN ratio was calculated as follows. First, both the Supplementary information QUEEN images were converted to signed 32-bit floating-point grayscale Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.230649.supplemental and then corrected for background using the rolling-ball algorithm or by subtracting the mean pixel values in an area outside the cells. Next, the References images were thresholded using the modified IsoData algorithm to set Barnett, J. A. and Entian, K. D. (2005). A history of research on yeasts 9: regulation background (non-thresholded) pixels to the Not a Number (NaN) value. The of sugar metabolism. Yeast 22, 835-894. pixel values of the ex410 image were divided by those of the ex480 image to Berg, J., Tymoczko, J. and Stryer, L. (2012). Biochemistry, 7th edn. New York: WH calculate the QUEEN ratio at each pixel. The ratio images were expressed in Freeman and Company. Journal of Cell Science

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