Reliable imaging of ATP in living budding and fission yeast

Takaine, Masak; Ueno, Masaru; Kitamura, Kenji; Imamura, Hiromi; Yoshida, Satoshi

doi:10.1242/jcs.230649

Cited by 39 publications

(63 citation statements)

References 43 publications

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“…We recently developed a reliable monitoring system for cytoplasmic ATP concentrations in living yeast cells using the ATP biosensor QUEEN (Takaine et al, 2019). We conducted a more detailed examination of ATP dynamics in wild-type and mutant yeast cells using this system in the present study.…”

Section: Resultsmentioning

confidence: 99%

“…Adenosine triphosphate (ATP) is the main energy currency used by all living organisms. Due to high demand, the turnover rate of ATP is estimated to be less than one minute in yeast and metazoans (Mortensen, Thaning et al, 2011, Takaine, Ueno et al, 2019). In addition to its role as energy currency, recent studies reported that ATP may influence the balance between the soluble and aggregated states of proteins, suggesting that proteostasis is maintained by energy-dependent chaperones and also by the property of ATP as a hydrotrope to solubilize proteins (Hayes, Peuchen et al, 2018, Patel, Malinovska et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…We recently established a reliable imaging technique to quantify intracellular ATP concentrations in single living yeast cells using the genetically encoded fluorescent ATP biosensor QUEEN (Yaginuma, Kawai et al, 2014), which enables long-term evaluations of ATP homeostasis in individual cells (Takaine et al, 2019). Our findings demonstrated that intracellular ATP concentrations did not vary within a yeast population grown in the same culture (Takaine et al, 2019), which was in contrast to the large variations observed in intracellular ATP concentrations within a bacterial cell population (Yaginuma et al, 2014). Moreover, intracellular ATP concentrations in individual living yeast cells were stably and robustly maintained at approximately 4 mM, irrespective of carbon sources and cell cycle stages, and temporal fluctuations in intracellular ATP concentrations were small (Takaine et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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High and stable ATP levels prevent aberrant intracellular protein aggregation

Takaine

Imamura

Yoshida

2019

Preprint

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23ATP is the main source of chemical energy in all life and is maintained at several millimolar 24 in eukaryotic cells. However, the mechanisms responsible for and physiological relevance of 25 this high and stable concentration of ATP remain unclear. We herein demonstrate that 26 AMP-activated protein kinase (AMPK) and adenylate kinase (ADK) cooperate to maintain 27 cellular ATP levels regardless of glucose concentrations. Single cell imaging of ATP-reduced 28 yeast mutants revealed that ATP concentrations in these mutants underwent stochastic and 29 transient depletion of ATP repeatedly, which induced the cytotoxic aggregation of 30 endogenous proteins and pathogenic proteins, such as huntingtin and α-synuclein. Moreover, 31 pharmacological elevations in ATP levels in an ATP-reduced mutant prevented the 32 accumulation of α-synuclein aggregates and its cytotoxicity. The removal of cytotoxic 33 aggregates depended on proteasomes, and proteasomal activity cooperated with AMPK or 34 ADK to resist proteotoxic stresses. The present results provide the first evidence to show that 35 cellular ATP homeostasis ensures proteostasis and revealed that stochastic fluctuations in 36 cellular ATP concentrations contribute to cytotoxic protein aggregation, implying that AMPK 37 and ADK are important factors that prevent proteinopathies, such as neurodegenerative 38 diseases. 39 40 2019); however, it currently remains unclear whether ATP-dependent protein 51 solubilization/desolubilization play any significant roles in cell physiology. 52 53 We recently established a reliable imaging technique to quantify intracellular ATP 54 concentrations in single living yeast cells using the genetically encoded fluorescent ATP 55 biosensor QUEEN (Yaginuma, Kawai et al., 2014), which enables long-term evaluations of 56 ATP homeostasis in individual cells (Takaine et al., 2019). Our findings demonstrated that 57 intracellular ATP concentrations did not vary within a yeast population grown in the same 58 culture (Takaine et al., 2019), which was in contrast to the large variations observed in 59 intracellular ATP concentrations within a bacterial cell population (Yaginuma et al., 2014). 60 Moreover, intracellular ATP concentrations in individual living yeast cells were stably and 61 robustly maintained at approximately 4 mM, irrespective of carbon sources and cell cycle 62 stages, and temporal fluctuations in intracellular ATP concentrations were small ( Takaine et 63 al., 2019). Based on these findings, we hypothesized that an exceptionally robust mechanism 64 may exist to precisely regulate ATP concentrations in eukaryotes. It also remains unclear why 65 ATP is stably maintained at a markedly higher concentration than the K m (Michaelis constant) 66 required for the enzymatic activity of almost all ATPases (Edelman, Blumenthal et al., 1987), 67 and the consequences associated with failed ATP homeostasis in living organisms have not 68 yet been elucidated. 69 70The most promising candidate regulator of ATP homeostasis is AMP-activated prote...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High and stable ATP levels prevent aberrant intracellular protein aggregation

Takaine

Imamura

Yoshida

2019

Preprint

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“…This observation that different levels of ATP may induce different cellular behaviours comes as increasing cell heterogeneity in ATP levels is being experimentally characterised. Intracellular ATP has been quantified to vary between 0.32-2.76mM in a bacterial population 39 , 3.7-3.9mM in HeLa cells 41 , 3-4mM in living yeast cells 40 and over a several-fold change occurs in different tissues of a plant 42 . The connection between energy variability and cellular decision-making has also been discussed 9/25 previously 30,54 , further supporting the hypothesis that energy diversity may modulate the number of regulatory states supported by a cell, playing an important causal role in inducing variability in cellular decision-making.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Energy Variability Modulates Cellular Decision-Making Capacity

Kerr

Jabbari

Johnston

2019

Preprint

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Cells are able to generate phenotypic diversity both during development and in response to stressful and changing environments, aiding survival. The biologically and medically vital process of a cell assuming a functionally important fate from a range of phenotypic possibilities can be thought of as a cell decision. To make these decisions, a cell relies on energy dependent pathways of signalling and expression. However, energy availability is often overlooked as a modulator of cellular decisionmaking. As cells can vary dramatically in energy availability, this limits our knowledge of how this key biological axis affects cell behaviour. Here, we consider the energy dependence of a highly generalisable decision-making regulatory network, and show that energy variability changes the sets of decisions a cell can make and the ease with which they can be made. Increasing intracellular energy levels can increase the number of stable phenotypes it can generate, corresponding to increased decision-making capacity. For this decision-making architecture, a cell with intracellular energy below a threshold is limited to a singular phenotype, potentially forcing the adoption of a specific cell fate. We suggest that common energetic differences between cells may explain some of the observed variability in cellular decision-making, and demonstrate the importance of considering energy levels in several diverse biological decision-making phenomena. endospores is a survival mechanism used by diverse genera including Bacillus and Clostridium 25 , contributing to foodborne disease and food spoilage 26,27 . The decision to become a persister cell (a phenotype more tolerant to external stress, including antibiotics), is made in several bacterial species 28-31 and can have a dramatic impact on the efficacy of treatments [32][33][34][35] .Many of the mechanisms behind cellular decision-making in eukaryotes and prokaryotes remain poorly understood. Regulatory networks, representing the interactions between genes that govern these decisions, are often used to summarise our knowledge 36 . Typically, edges in a schematic network illustrate processes such as transcription and translation and nodes represent genes. However, this coarse-graining can often omit a substantial amount of important detail. In particular, the fact that the processes represented by these edges are energy dependent (Figure 1) is rarely considered. Transcription and translation require a substantial ATP budget 37, 38 , so there exists a core energy dependence in the dynamics of gene regulatory networks, potentially affecting the decisions supported by a given cell.This energy dependence is important because different cells, particularly in microbiology, can have substantially different levels of available energy. Energy variability has been observed within genetically-identical Escherichia coli bacteria cells in a population, where absolute concentrations of intracellular ATP were spread over at least half an order of magnitude, in a skewed distribution around 1.54 ± 1...

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“…However, continuous measurements of ATP, and hence glycolytic startup dynamics, have not been studied at a single-cell level. In recent years several fluorescence-based biosensors for in vivo monitoring of ATP in single cells have been developed, including the AT1.03 and the QUEEN sensor 23,24 . However, these sensors use fluorescent proteins (FPs) that are pH-sensitive in the physiological range where the intracellular pH of budding yeast operates 17,[25][26][27][28][29] .…”

Section: Introductionmentioning

confidence: 99%

An improved ATP FRET sensor for yeast shows heterogeneity during nutrient transitions

Botman

Heerden

Teusink

2019

Preprint

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Adenosine 5-triphosphate (ATP) is the main free energy carrier in metabolism. In budding yeast, shifts to glucose-rich conditions cause dynamic changes in ATP levels, but it is unclear how heterogeneous these dynamics are at the single-cell level. Furthermore, pH also changes and affects readout of fluorescence-based biosensors for single-cell measurements. To measure ATP changes reliably in single yeast cells, we developed yAT1.03, an adapted version of the AT1.03 ATP biosensor, that is pHinsensitive. We show that pregrowth conditions largely affect ATP dynamics during transitions. Moreover, single-cell analyses showed a large variety in ATP responses, which implies large differences of glycolytic startup between individual cells. We found three clusters of dynamic responses, and show that a small subpopulation of wild type cells reached an imbalanced state during glycolytic startup, characterised by low ATP levels. These results confirm the need for new tools to study dynamic responses of individual cells in dynamic environments.

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Reliable imaging of ATP in living budding and fission yeast

Cited by 39 publications

References 43 publications

High and stable ATP levels prevent aberrant intracellular protein aggregation

High and stable ATP levels prevent aberrant intracellular protein aggregation

Intracellular Energy Variability Modulates Cellular Decision-Making Capacity

An improved ATP FRET sensor for yeast shows heterogeneity during nutrient transitions

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