Redox biosensors in a context of multiparameter imaging

Kostyuk, Alexander I.; Panova, Anastasiya S.; Bilan, Dmitry S.; Belousov, Vsevolod V.

doi:10.1016/j.freeradbiomed.2018.04.004

Cited by 30 publications

(14 citation statements)

References 219 publications

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“…The HyPer family of fluorescence probes has been developed for the selective detection of H 2 O 2 ( 3 , 4 , 38 , 54 ). We have employed vectors encoding mito-HyPer and mito-SypHer (kindly provided by Dr. Enyedi, Semmelweis University, Hungary).…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio

Plecitá–Hlavatá

Engstová

Holendová

et al. 2020

Antioxidants & Redox Signaling

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Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H 2 O 2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH F, indicated its *20% decrease. Matrix NAD + F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD + F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH F (iNAP1 fluorescence monitoring) at the expense of matrix NADH F , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13 Cincorporation from 13 C-L-glutamine into 13 C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH F (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I F site contribution, but a lower superoxide fraction ascribed to the Complex III site III Qo. Thus, the diminished matrix NADH F /NAD + F decreased Complex I flavin site I F superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic b cells on GSIS, due to the decreasing matrix NADH F /NAD + F (NADPH F /NADP + F) at increasing cytosolic NADPH F levels. The developed innovative methods enable real-time NADH/NAD + and NADPH/ NADP + monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic b cells. Antioxid. Redox Signal. 33, 789-815.

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Section: Methodsmentioning

confidence: 99%

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio

Plecitá–Hlavatá

Engstová

Holendová

et al. 2020

Antioxidants & Redox Signaling

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“…This fact imposes a significant limitation on the use of these tools in the multiparameter imaging mode in combination with other spectrally distinct fluorophores. This approach allows the simultaneous monitoring of either different activities within the same cellular organelle or the same parameter in different organelles of the cell or even in different types of cells within the tissue [25]. As a result, new spectral versions of roFPs are being developed.…”

Section: Introductionmentioning

confidence: 99%

Red fluorescent redox-sensitive biosensor Grx1-roCherry

et al. 2019

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Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of − 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local H2O2 production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging.

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“…Progress in studying hypoxic/ischemic injury of the CNS only began when the following methods became available: powerful platforms for live imaging that were primarily reliant on optical methods (fluorescence, confocal and two-photon microscopy, and photoacoustic tomography [15]); nuclear technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) [16,17], and a plethora of chemogenic and genetically encoded fluorescent indicators for probing a variety of biologically significant ions, signaling molecules, and metabolites, including those involved in redox regulation [18][19][20][21][22]. Although they are the principal tools used for clinical and diagnostic purposes, nuclear technologies provide insufficient spatiotemporal resolution of brain functioning at a cellular level.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, optical imaging combined with the use of fluorescent indicators is a fundamental tool used in basic research; this method enables the noninvasive detection of various intracellular metabolites or signaling molecules in real time with single-cell or even subcellular resolution. Genetically encoded biosensors have many advantages over other types of fluorescent probes (reviewed in [ 20 , 21 , 23 , 24 , 25 , 26 ]) and have allowed unmatched opportunities for studying hypoxic/ischemic injury of the brain. Genetically encoded biosensors may be expressed in a spatiotemporally defined manner in specific cell types or subcellular compartments.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

et al. 2020

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Hypoxia is characterized by low oxygen content in the tissues. The central nervous system (CNS) is highly vulnerable to a lack of oxygen. Prolonged hypoxia leads to the death of brain cells, which underlies the development of many pathological conditions. Despite the relevance of the topic, different approaches used to study the molecular mechanisms of hypoxia have many limitations. One promising lead is the use of various genetically encoded tools that allow for the observation of intracellular parameters in living systems. In the first part of this review, we provide the classification of oxygen/hypoxia reporters as well as describe other genetically encoded reporters for various metabolic and redox parameters that could be implemented in hypoxia studies. In the second part, we discuss the advantages and disadvantages of the primary hypoxia model systems and highlight inspiring examples of research in which these experimental settings were combined with genetically encoded reporters.

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Redox biosensors in a context of multiparameter imaging

Cited by 30 publications

References 219 publications

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio

Red fluorescent redox-sensitive biosensor Grx1-roCherry

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

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Redox biosensors in a context of multiparameter imaging

Cited by 30 publications

References 219 publications

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+Ratio

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+Ratio

Red fluorescent redox-sensitive biosensor Grx1-roCherry

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

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Product

Resources

About

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio

Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD⁺Ratio