RGB‐Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

Arai, Soichi; Kriszt, Rókus; Harada, Kazuki; Looi, Liang Sheng; Matsuda, Shogo; Wongso, Devina; Suo, Satoshi; Ishiura, Shoichi; Tseng, Yu–Hua; Raghunath, Michael; Ito, Toshiro; Tsuboi, Takashi; Kitaguchi, Tetsuya

doi:10.1002/anie.201804304

Cited by 97 publications

(91 citation statements)

References 28 publications

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“…AT and ATY (but has been reported for ATP sensors using a single fluorophore; Arai et al, 2018), both sensors could even be recorded within a single cell. Values for k D and n H need to be obtained from other experimental systems, e.g., from measurements of the purified sensor protein (Imamura et al, 2009), assuming that these values can be applied to the indicators in the cytosolic environment of the cells of interest ( Table 1).…”

Section: Properties Assumptions and Limitations Of The Dual Nanosensmentioning

confidence: 99%

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

Köhler

Schmidt

Fülle

et al. 2020

Front. Cell. Neurosci.

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Adenosine triphosphate (ATP) is the central energy carrier of all cells and knowledge on the dynamics of the concentration of ATP ([ATP]) provides important insights into the energetic state of a cell. Several genetically encoded fluorescent nanosensors for ATP were developed, which allow following the cytosolic [ATP] at high spatial and temporal resolution using fluorescence microscopy. However, to calibrate the fluorescent signal to [ATP] has remained challenging. To estimate basal cytosolic [ATP] ([ATP] 0) in astrocytes, we here took advantage of two ATP nanosensors of the ATeam-family (ATeam1.03; ATeam1.03YEMK) with different affinities for ATP. Altering [ATP] by external stimuli resulted in characteristic pairs of signal changes of both nanosensors, which depend on [ATP] 0. Using this dual nanosensor strategy and epifluorescence microscopy, [ATP] 0 was estimated to be around 1.5 mM in primary cultures of cortical astrocytes from mice. Furthermore, in astrocytes in acutely isolated cortical slices from mice expressing both nanosensors after stereotactic injection of AAV-vectors, 2-photon microscopy revealed [ATP] 0 of 0.7 mM to 1.3 mM. Finally, the change in [ATP] induced in the cytosol of cultured cortical astrocytes by application of azide, glutamate, and an increased extracellular concentration of K + were calculated as −0.50 mM, −0.16 mM, and 0.07 mM, respectively. In summary, the dual nanosensor approach adds another option for determining the concentration of [ATP] to the increasing toolbox of fluorescent nanosensors for metabolites. This approach can also be applied to other metabolites when two sensors with different binding properties are available.

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Section: Properties Assumptions and Limitations Of The Dual Nanosensmentioning

confidence: 99%

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

Köhler

Schmidt

Fülle

et al. 2020

Front. Cell. Neurosci.

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“…ATPOS has at least four advantages for in vivo visualization of extracellular ATP. First, ATPOS has a very high affinity for ATP; its apparent dissociation constant is ~150 nM, while those of previously reported fluorescent ATP sensors, such as ATeam, QUEEN and iATPSnFR, are in the range of several micromolar to several millimolar ( Arai et al, 2018 ; Conley et al, 2017 ; Imamura et al, 2009 ; Lobas et al, 2019 ; Yaginuma et al, 2015 ). The low affinity of the conventional sensors makes them unsuitable for in vivo extracellular ATP imaging.…”

Section: Discussionmentioning

confidence: 93%

“…On the other hand, imaging techniques using fluorescent sensors can provide excellent spatiotemporal resolution ( Giepmans et al, 2006 ), and a variety of fluorescent ATP sensors based on fluorescent proteins have been developed ( Arai et al, 2018 ; Berg et al, 2009 ; Imamura et al, 2009 ; Lobas et al, 2019 ; Tantama et al, 2013 ; Yaginuma et al, 2015 ). These genetically encoded ATP sensors can visualize intracellular ATP dynamics, and have facilitated our understanding of ATP signaling inside cells ( Depaoli et al, 2018 ; Zala et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

Kitajima

Takikawa

Sekiya

et al. 2020

eLife

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Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

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“…Neuronal ATP levels increased simultaneously with the level of the arousal of animals, whereas they profoundly decreased in an REM sleepspecific manner. These in vivo observations were achieved by genetically encoded ATP sensors recently developed and adopted 12,14,23,[41][42][43][44][45][46][47] , as well as fiber photometry and wide-field microscopy, which was originally used for in vivo monitoring of neuronal calcium activities 19,48 . Our data demonstrate the superiority of these techniques over previously used, conventional methodologies such as chemiluminescence imaging by luciferase 49 , which lack temporal and spatial resolutions, are sensitive to a high pH, and importantly, are restricted to in vitro/ ex vivo application.…”

Section: Discussionmentioning

confidence: 99%

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep–wake states

et al. 2020

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Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5’-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

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RGB‐Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

Cited by 97 publications

References 28 publications

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep–wake states

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