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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,*

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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,* © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs230649. doi:10.1242/jcs.230649 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 1 TOOLS AND RESOURCES Journal of Cell Science (2019) 132, jcs230649. doi:10.1242/jcs.230649 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 2 TOOLS AND RESOURCES Journal of Cell Science (2019) 132, jcs230649. doi:10.1242/jcs.230649 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.
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