MITOCHONDRIA: HUBS OF CELLULAR SIGNALING, ENERGETICS AND REDOX BALANCE EDITED BY : Miguel A. Aon and Amadou K. S. Camara PUBLISHED IN : Frontiers in Physiology 1 July 2017 | Mitochondria, hubs of cellular signaling-energetics Frontiers in Physiology Frontiers Copyright Statement © Copyright 2007-2017 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA (“Frontiers”) or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers. The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. 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Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: researchtopics@frontiersin.org 2 July 2017 | Mitochondria, hubs of cellular signaling-energetics Frontiers in Physiology MITOCHONDRIA: HUBS OF CELLULAR SIGNALING, ENERGETICS AND REDOX BALANCE Mitochondria are dynamic organelles structurally and functionally connected with other cellular compartments. The mitochondrial network continuously undergoes fusion and fission. Close proximity and physical interaction between mitochondria and ER influences homeostasis of both organelles. Other interactions include association of mitochondria with lipid droplets or modulation of nuclear transcriptional response through retrograde signaling. Figure by Soni Deshwal and Nina Kaludercic. Figure was taken and modified from Kaludercic N, Deshwal S and Di Lisa F (2014) Reactive oxygen species and redox compartmentalization. Front. Physiol. 5:285. doi:10.3389/fphys.2014.00285 Topic Editors: Miguel A. Aon, Johns Hopkins University and National Institute on Aging/NIH, United States Amadou K. S. Camara, Medical College of Wisconsin, United States 3 July 2017 | Mitochondria, hubs of cellular signaling-energetics Frontiers in Physiology Poised at the convergence of most catabolic and anabolic pathways, mitochondria are the center of heterotrophic aerobic life, representing a hub in the overall metabolic network of cells. The energetic functions performed by mitochondria face the unavoidable redox hurdle of handling huge amounts of oxygen while keeping its own as well as the cellular redox environment under control. Reactive oxygen species (ROS) are produced in the respiratory chain as a result of the energy supplying function of mitochondria. Originally considered an unavoidable by-product of oxi- dative phosphorylation, ROS have become crucial signaling molecules when their levels are kept within physiological range. This occurs when their production and scavenging are balanced within mitochondria and cells. Mitochondria-generated hydrogen peroxide can act as a signaling molecule within mitochon- dria or in the cytoplasm, affecting multiple networks that control, for example, cell cycle, stress response, cell migration and adhesion, energy metabolism, redox balance, cell contraction, and ion channels. However, under pathophysiological conditions, excessive ROS levels can happen due to either overproduction, overwhelming of antioxidant defenses, or both. Under oxidative stress, detrimental effects of ROS include oxidation of protein, lipids, and nucleic acids; mito- chondrial depolarization and calcium overload; and cell-wide oscillations mediated by ROS- induced ROS release mechanisms. Mitochondrial dysfunction is central in the pathogenesis of numerous human maladies including cardiomyopathies and neurodegeneration. Diseases characterized by altered nutrient metabolism, such as diabetes and cancer, exhibit elevated ROS levels. These may contribute to pathogenesis by increasing DNA mutation, affecting regulatory signaling and transcription, and promoting inflammation. Under metabolic stress, several ionic channels present in the inner and outer mitochondrial membranes can have pro-life and -death effects. In the present E-book, based on the Frontiers Research Topic entitled: “Mitochondria: Hubs of cellular signaling, energetics and redox balance”, we address one of the fundamental questions that the field of ROS biology faces today: how do mitochondria accomplish a reliable energy provision and at the same time keep ROS levels within physiological, non-harming, limits but crucial for cellular signaling function? Additionally, and within the perspective of mitochondria as signaling-energetic hubs in the extensive cellular metabolic network, we ask how can their collective dynamics scale from the subcellular to the cellular, tissue and organ levels to affect function in health and disease. Citation: Aon, M. A., Camara, A. K. S., eds. (2017). Mitochondria: Hubs of Cellular Signaling, Energetics and Redox Balance. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-239-2 4 July 2017 | Mitochondria, hubs of cellular signaling-energetics Frontiers in Physiology Table of Contents Editorial 06 Mitochondria: hubs of cellular signaling, energetics and redox balance. A rich, vibrant, and diverse landscape of mitochondrial research Miguel A. Aon and Amadou K. S. Camara I. Compartmentation of energetic and redox functions in subcellular networks 09 Reactive oxygen species and redox compartmentalization Nina Kaludercic, Soni Deshwal and Fabio Di Lisa 24 The location of energetic compartments affects energetic communication in cardiomyocytes Rikke Birkedal, Martin Laasmaa and Marko Vendelin 33 Cardiac mitochondria exhibit dynamic functional clustering Felix T. Kurz, Miguel A. Aon, Brian O’Rourke and Antonis A. Armoundas 41 Complex oscillatory redox dynamics with signaling potential at the edge between normal and pathological mitochondrial function Jackelyn M. Kembro, Sonia Cortassa and Miguel A. Aon 52 Mitochondrial and cellular mechanisms for managing lipid excess Miguel A. Aon, Niraj Bhatt and Sonia C. Cortassa II. Signaling and mitochondrial function 65 Bcl-xL in neuroprotection and plasticity Elizabeth A. Jonas, George A. Porter and Kambiz N. Alavian 75 Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria Kyriakos N. Papanicolaou, Brian O’Rourke and D. Brian Foster 97 Necroptosis: is there a role for mitochondria? Kurt D. Marshall and Christopher P . Baines 102 Role of b -hydroxybutyrate, its polymer poly- b -hydroxybutyrate and inorganic polyphosphate in mammalian health and disease Elena N. Dedkova and Lothar A. Blatter III. The impact of mitochondrial and cellular redox balance on disease 124 The “Goldilocks Zone” from a redox perspective—Adaptive vs. deleterious responses to oxidative stress in striated muscle Rick J. Alleman, Lalage A. Katunga, Margaret A. M. Nelson, David A. Brown and Ethan J. Anderson 5 July 2017 | Mitochondria, hubs of cellular signaling-energetics Frontiers in Physiology 144 The impact of age-related dysregulation of the angiotensin system on mitochondrial redox balance Ramya Vajapey, David Rini, Jeremy Walston and Peter Abadir 161 Hypertrophic cardiomyopathy: a heart in need of an energy bar? Styliani Vakrou and M. Roselle Abraham 169 A deficiency of apoptosis inducing factor (AIF) in Harlequin mouse heart mitochondria paradoxically reduces ROS generation during ischemia-reperfusion Qun Chen, Karol Szczepanek, Ying Hu, Jeremy Thompson and Edward J. Lesnefsky 179 Differential effects of buffer pH on Ca 2+-induced ROS emission with inhibited mitochondrial complexes I and III Daniel P . Lindsay, Amadou K. S. Camara, David F . Stowe, Ryan Lubbe and Mohammed Aldakkak 189 Functional crosstalk between the mitochondrial PTP and K ATP channels determine arrhythmic vulnerability to oxidative stress Chaoqin Xie, Justin Kauffman and Fadi G. Akar IV. Interventions leading to protection of mitochondrial function 200 Mitochondrial targets for volatile anesthetics against cardiac ischemia-reperfusion injury Bhawana Agarwal, David F . Stowe, Ranjan K. Dash, Zeljko J. Bosnjak and Amadou K. S. Camara 217 Far red/near infrared light-induced protection against cardiac ischemia and reperfusion injury remains intact under diabetic conditions and is independent of nitric oxide synthase Agnes Keszler, Garth Brandal, Shelley Baumgardt, Zhi-Dong Ge, Phillip F . Pratt, Matthias L. Riess and Martin Bienengraeber V. Research topic highlight comments 224 Blue LEDs get the Nobel Prize while Red LEDs are poised to save lives Basil S. Karam and Fadi G. Akar 226 The calcium-ROS-pH triangle and mitochondrial permeability transition: challenges to mimic cardiac ischemia-reperfusion Sabzali Javadov EDITORIAL published: 26 March 2015 doi: 10.3389/fphys.2015.00094 Frontiers in Physiology | www.frontiersin.org March 2015 | Volume 6 | Article 94 | Edited and reviewed by: Paolo Bernardi, University of Padova, Italy *Correspondence: Miguel A. Aon, maon1@jhmi.edu Specialty section: This article was submitted to Mitochondrial Research, a section of the journal Frontiers in Physiology Received: 03 March 2015 Accepted: 12 March 2015 Published: 26 March 2015 Citation: Aon MA and Camara AKS (2015) Mitochondria: hubs of cellular signaling, energetics and redox balance. A rich, vibrant, and diverse landscape of mitochondrial research. Front. Physiol. 6:94. doi: 10.3389/fphys.2015.00094 Mitochondria: hubs of cellular signaling, energetics and redox balance. A rich, vibrant, and diverse landscape of mitochondrial research Miguel A. Aon 1 * and Amadou K. S. Camara 2 1 Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD, USA, 2 Department of Anesthesiology and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI, USA Keywords: energetic-redox compartmentation, posttranslational modification, apoptosis inducing factor, beta oxidation, permeability transition pore, infrared light heart protection, neuroprotection, mitochondrial network clustering Mitochondria have become the cornerstone of cellular biology, opening new frontiers in health and disease. Poised at the convergence of most catabolic and anabolic pathways, mitochondria consti- tute the center of heterotrophic aerobic life, processing and generating key metabolites that feed into pathways leading to growth, division, and signaling. As such, mitochondria play the role of hubs in the overall metabolic network; thus their failure risks the collapse of most crucial cellular functions. The protagonist role of mitochondria is underscored by the existence of tightly regu- lated cellular processes that include autophagy/mitophagy and metabolically tuned morphological changes induced by fusion-fission dynamics. Mitochondria are also involved in a myriad of sig- naling cascades regulating cell survival vs. death. They provide the energy for the cell, a hub for biosynthetic processes and they contain a self-destructive arsenal of apoptotogenic factors that can be unleashed to promote apoptotic signaling. Consequently, it is no wonder that mitochondrial dysfunction is implicated in the aging phenomenon, and in numerous human maladies includ- ing metabolic disorders, cardiomyopathies, and neurodegeneration. Indeed, diseases characterized by altered gene-nutrient interactions, such as diabetes and cancer, exhibit elevated levels of reac- tive oxygen species (ROS) of which mitochondria are a major source. Therefore, mitochondrial dysfunction and the resulting oxidative stress are central in these and other human pathologies. Excess ROS induced by oxidative stress are specifically known to contribute to these pathogeneses in part by increasing mitochondrial DNA mutations that, via retrograde signaling, affects nuclear gene expression, ultimately modulating gene transcription, protein translation, post-translational modifications, and cellular signaling. For this Research Topic, our initial focus was on the younger generation of researchers working on mitochondria, believing both in their responsibility to take the next steps in this field, confident in their ability to take mitochondrial research to new and exhilarating heights. In this endeavor, 18 papers, including a Commentary to one of the contributions, reveal an exciting, broad scope of sub- jects involving mitochondrial research that exploit a variety of novel methods, insights, and ideas. The potential implications of mitochondria at the crossroad of cellular injury and therapeutics are also addressed. Fresh insights into actively investigated areas affecting mitochondrial/cellular redox balance are highlighted in a series of review articles, as applied to the etiology of heart disease and insulin resistance (Alleman et al., 2014), and the role of the renin angiotensin system (Vajapey et al., 2014). The role of cellular redox compartmentalization (matrix vs. extra-matrix) in leading mitochondrial function from normal to pathological conditions (Kembro et al., 2014), 6 Aon and Camara Mitochondria, hubs of cellular signaling-energetics and the functional significance of the differential redox status exhibited by subcellular organelles (likely lysosomes, peroxisomes, endoplasmic reticulum, and nuclei) apart from mitochondria (Kaludercic et al., 2014) are also reported. A review contribution by Birkedal et al. (2014), and an origi- nal research article by Kurz et al. (2014) highlight the use of new conceptual and computational tools to address intracellular ener- getic compartmentalization and mitochondrial network orga- nization as essential components of the coordinated energetic response during the cardiac systole-diastole cycle. Hypertrophic cardiomyopathy, the most common inherited cardiac disease, is analyzed in a review by Vakrou and Abraham (2014) and defined as a metabolic disease in which mitochondrial function plays a relevant role. A novel experiment on the protective role of infrared light against cardiac injury elicited under ischemia and reperfusion (IR) is presented in Keszler et al. (2014) and highlighted in a Commentary article by Karam and Akar (2014). In another study, Chen et al. (2014) report on the flavin-NADH-containing apoptosis-inducing factor (AIF), found in mitochondria and required for optimal respiratory function. AIF acts as a stimula- tor of ROS-mediated cell death during IR in the Harlequin mouse model that expresses AIF in reduced amounts. Additional studies using intact heart, analyze the role of mitochondrial ion channels in arrhythmic propensity under oxidative stress, during IR injury (Xie et al., 2014). Whereas low levels of ROS can serve as criti- cal signaling molecules, excess ROS are implicated in IR injury. Lindsay et al. (2015) report on specific electron transport chain (ETC) complexes that are responsible for ROS generation under conditions that may prevail during prolonged ischemia. These include different substrate utilization, excessive mitochondrial calcium load and change in pH that culminate in mitochondrial permeability transition pore opening and ROS emission. Signaling aspects related to mitochondrial activity also had a prominent contribution in this Research Topic. Protective strate- gies of mitochondrial energetic function in neurons based on Bcl-xL, a member of the anti-apoptotic Bcl-2 protein family, and directed to prevent cell death via mitochondrial permeability transition pore opening and outer membrane permeabilization, are addressed by Jonas et al. (2014). In a review article Papanico- laou et al. (2014) discussed the new emerging family of mitochon- drial proteins that are post-translationally modified via direct reaction of lysine residues with activated thioester coenzyme A intermediates, and their functional impact on mitochondrial sir- tuins. In a Perspective article, the role of mitochondrial dysfunc- tion and the molecular mechanisms participating in necroptosis, a form of necrosis, are critically examined by Marshall and Baines (2014). A relatively new area of research is covered by Ded- kova and Blatter (2014) who extensively review the role of the ketone body β -hydroxybutyrate, its polymer poly- β -hydroxybutyrate, and inorganic polyphosphate, in diverse cellular functions, including mitochondrial ion transport, ener- getics and activation of mitochondrial permeability transition by polyphosphate. Detailed mechanisms of potential cardioprotection by volatile anesthetics to target mitochondrial channels/transporters and ETC complexes are described in Agarwal et al. (2014). Recent emergent molecular processes mediating physical-metabolic interactions between lipid droplets and mitochondria and their potential impact on fatty acid oxidation and generation of signal- ing ROS are explored in the contribution by Aon et al. (2014). Overall, the work in this Research Topic exemplifies many situations in which the morphological and functional behav- ior of mitochondria is sensitively tuned to the changing cel- lular energetic-redox status. The Research Topic sought clues about the underlying mechanisms that allow mitochondria to accomplish their energetic function while at the same time con- fronted with the unavoidable redox hurdle of processing huge amounts of oxygen as well as preserving the cellular redox envi- ronment. Since these conditions could be antithetical with the organelle’s survival, many contributions shed light on the fact that mitochondrial targeted approaches to treat diseases could be a harbinger for their protection and concomitantly cytoprotection in different pathologies. In this case, we have come full circle: the protector, the mitochondrion, has to be protected. References Agarwal, B., Stowe, D. F., Dash, R. K., Bosnjak, Z. J., and Camara, A. K. (2014). Mitochondrial targets for volatile anesthetics against cardiac ischemia- reperfusion injury. Front. Physiol. 5:341. doi: 10.3389/fphys.2014.00341 Alleman, R. J., Katunga, L. A., Nelson, M. A., Brown, D. A., and Anderson, E. J. (2014). The “Goldilocks Zone” from a redox perspective-Adaptive vs. deleteri- ous responses to oxidative stress in striated muscle . Front. Physiol. 5:358. doi: 10.3389/fphys.2014.00358 Aon, M. A., Bhatt, N., and Cortassa, S. C. (2014). Mitochondrial and cel- lular mechanisms for managing lipid excess. Front. Physiol. 5:282. doi: 10.3389/fphys.2014.00282 Birkedal, R., Laasmaa, M., and Vendelin, M. (2014). The location of energetic com- partments affects energetic communication in cardiomyocytes. Front. Physiol. 5:376. doi: 10.3389/fphys.2014.00376 Chen, Q., Szczepanek, K., Hu, Y., Thompson, J., and Lesnefsky, E. J. (2014). A deficiency of apoptosis inducing factor (AIF) in Harlequin mouse heart mito- chondria paradoxically reduces ROS generation during ischemia-reperfusion. Front. Physiol. 5:271. doi: 10.3389/fphys.2014.00271 Dedkova, E. N., and Blatter, L. A. (2014). Role of beta-hydroxybutyrate, its polymer poly-beta-hydroxybutyrate and inorganic polyphosphate in mammalian health and disease. Front. Physiol. 5:260. doi: 10.3389/fphys.2014.00260 Jonas, E. A., Porter, G. A., and Alavian, K. N. (2014). Bcl-xL in neuroprotection and plasticity. Front. Physiol. 5:355. doi: 10.3389/fphys.2014.00355 Kaludercic, N., Deshwal, S., and Di Lisa, F. (2014). Reactive oxygen species and redox compartmentalization. Front. Physiol. 5:285. doi: 10.3389/fphys.2014.00285 Karam, B. S., and Akar, F. G. (2014). Blue LEDs get the Nobel Prize while Red LEDs are poised to save lives. Front. Physiol. 5:443. doi: 10.3389/fphys.2014.00443 Kembro, J. M., Cortassa, S., and Aon, M. A. (2014). Complex oscillatory redox dynamics with signaling potential at the edge between normal and patho- logical mitochondrial function. Front. Physiol. 5:257. doi: 10.3389/fphys.2014. 00257 Keszler, A., Brandal, G., Baumgardt, S., Ge, Z. D., Pratt, P. F., Riess, M. L., et al. (2014). Far red/near infrared light-induced protection against car- diac ischemia and reperfusion injury remains intact under diabetic condi- tions and is independent of nitric oxide synthase. Front. Physiol. 5:305. doi: 10.3389/fphys.2014.00305 Frontiers in Physiology | www.frontiersin.org March 2015 | Volume 6 | Article 94 | 7 Aon and Camara Mitochondria, hubs of cellular signaling-energetics Kurz, F. T., Aon, M. A., O’Rourke, B., and Armoundas, A. A. (2014). Cardiac mitochondria exhibit dynamic functional clustering. Front. Physiol. 5:329. doi: 10.3389/fphys.2014.00329 Lindsay, D. P., Camara, A. K., Stowe, D. F., Lubbe, R., and Aldakkak, M. (2015). Differential effects of buffer pH on Ca2+-induced ROS emission with inhibited mitochondrial complex I and III. Front. Physiol. 6:58. doi: 10.3389/fphys.2015.00058 Marshall, K. D., and Baines, C. P. (2014). Necroptosis: is there a role for mitochon- dria? Front. Physiol. 5:323. doi: 10.3389/fphys.2014.00323 Papanicolaou, K. N., O’Rourke, B., and Foster, D. B. (2014). Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria. Front. Physiol. 5:301. doi: 10.3389/fphys.2014.00301 Vajapey, R., Rini, D., Walston, J., and Abadir, P. (2014). The impact of age-related dysregulation of the angiotensin system on mitochondrial redox balance. Front. Physiol. 5:439. doi: 10.3389/fphys.2014.00439 Vakrou, S., and Abraham, M. R. (2014). Hypertrophic cardiomyopathy: a heart in need of an energy bar? Front. Physiol. 5:309. doi: 10.3389/fphys.2014.00309 Xie, C., Kauffman, J., and Akar, F. G. (2014). Functional crosstalk between the mitochondrial PTP and KATP channels determine arrhythmic vulner- ability to oxidative stress. Front. Physiol. 5:264. doi: 10.3389/fphys.2014. 00264 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2015 Aon and Camara. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Physiology | www.frontiersin.org March 2015 | Volume 6 | Article 94 | 8 REVIEW ARTICLE published: 12 August 2014 doi: 10.3389/fphys.2014.00285 Reactive oxygen species and redox compartmentalization Nina Kaludercic 1 *, Soni Deshwal 2 and Fabio Di Lisa 1,2 1 Neuroscience Institute, National Research Council of Italy (CNR), Padova, Italy 2 Department of Biomedical Sciences, University of Padova, Padova, Italy Edited by: Miguel A. Aon, Johns Hopkins University School of Medicine, USA Reviewed by: David Lloyd, Cardiff University, UK Amadou K. S. Camara, Medical College of Wisconsin, USA *Correspondence: Nina Kaludercic, Neuroscience Institute, National Research Council of Italy (CNR), Viale G. Colombo 3, 35131 Padova, Italy e-mail: nina.kaludercic@unipd.it Reactive oxygen species (ROS) formation and signaling are of major importance and regulate a number of processes in physiological conditions. A disruption in redox status regulation, however, has been associated with numerous pathological conditions. In recent years it has become increasingly clear that oxidative and reductive modifications are confined in a spatio-temporal manner. This makes ROS signaling similar to that of Ca 2 + or other second messengers. Some subcellular compartments are more oxidizing (such as lysosomes or peroxisomes) whereas others are more reducing (mitochondria, nuclei). Moreover, although more reducing, mitochondria are especially susceptible to oxidation, most likely due to the high number of exposed thiols present in that compartment. Recent advances in the development of redox probes allow specific measurement of defined ROS in different cellular compartments in intact living cells or organisms. The availability of these tools now allows simultaneous spatio-temporal measurements and correlation between ROS generation and organelle and/or cellular function. The study of ROS compartmentalization and microdomains will help elucidate their role in physiology and disease. Here we will examine redox probes currently available and how ROS generation may vary between subcellular compartments. Furthermore, we will discuss ROS compartmentalization in physiological and pathological conditions focusing our attention on mitochondria, since their vulnerability to oxidative stress is likely at the basis of several diseases. Keywords: reactive oxygen species, compartmentalization, mitochondria, oxidative stress, redox signaling INTRODUCTION Reactive oxygen species (ROS) formation and redox signaling are well known to play a major role in physiology as well as in a variety of pathologies. For instance, in the heart, cardiomyocyte differen- tiation, and excitation-contraction coupling are under tight redox control (Burgoyne et al., 2012; Steinberg, 2013). On the other hand, cardiac pathologies, such as ischemia/reperfusion injury, heart failure, and arrhythmias can be prevented or blocked by inhibiting specific processes that result in ROS generation in sev- eral experimental models (Takimoto and Kass, 2007; Youn et al., 2013; Anderson et al., 2014; Kaludercic et al., 2014b). Thus, it appears that pro-oxidant generation and antioxidant defense need to be tightly regulated (Chance et al., 1979). Indeed, dis- ruption of redox signaling and control, and imbalance in favor of pro-oxidant species is defined oxidative stress, term first coined in 1985 (Sies, 1985; Sies and Cadenas, 1985; Jones, 2006). Conversely from pathological modifications (Chance et al., 1979; Powers and Jackson, 2008), it appears that physiological redox signaling is characterized by reversible oxido-reductive modifications, con- fined both spatially and temporally in subcellular compartments and microdomains. To exert their effects, ROS have to induce a reversible change that results in the modification of protein activity. The first step is the single-electron oxidation of a thiol to a thiyl radical, which can then react to form disulphide bonds with glutathione (GSH) or with another protein thiol (Wardman and Von Sonntag, 1995; Collins et al., 2012). Nevertheless, thiols can be further oxidized by ROS and result in higher oxidation states of sulfur (Steinberg, 2013). Such changes have limited or no reversibil- ity under biological conditions (Jones and Go, 2010; Steinberg, 2013). Quantification of thiol oxidation on cellular and subcellular levels has shown that thiol/disulphide couples such as GSH and thioredoxin (Trx) are maintained at stable values and are not in equilibrium relative to each other in different organelles (Go and Jones, 2008; Jones and Go, 2010). This suggests that redox status as a consequence of ROS production is not necessarily a global imbalance of oxidative and reductive processes, but rather that thiol oxidation in different cellular compartments serves as means for cell signaling, protein trafficking and regulation of enzyme, receptor, transporter and transcription factor activity (Balaban et al., 2005; D’Autreaux and Toledano, 2007). This consideration, termed the “redox hypothesis,” postulates that oxidizable thiols are control elements organized in redox circuits that are physi- cally and kinetically separated so that they are highly responsive and can function independently to regulate different biological processes (Jones and Go, 2010). However, upon a certain thresh- old in ROS formation, these circuits can be disrupted. Indeed, the occurrence of oxidative stress overwhelms the cellular antioxidant defense and results in lack of control over redox signaling mech- anisms. These concepts can now be validated employing new redox sensors that allow dynamic and compartmentalized ROS www.frontiersin.org August 2014 | Volume 5 | Article 285 | 9 Kaludercic et al. ROS compartmentalization measurements and their correlation with organelle/cell function and viability. Thus, ROS generation within specific subcellular compart- ments and their redox status appear to be of major importance for understanding cell pathophysiology. Recently, new methods for the study of redox compartmentalization have become available. This is a rapidly growing field that led to the development of, and was then contributed by, probes that now permit observation of rapid redox changes in real time and with single organelle resolu- tion not only in live cells, but also in living animals (Woolley et al., 2013; Ezerina et al., 2014; Lukyanov and Belousov, 2014). Here we will review the tools currently available for the measurement of ROS and redox potential within single organelles and discuss the data available so far on ROS compartmentalization in phys- iological and pathological conditions focusing our attention on mitochondria as the major source and target of ROS. TOOLS TO STUDY COMPARTMENT REDOX STATUS AND ROS FORMATION In order to study the relationship between ROS formation and cell (dys)function, it is necessary to define which species are produced, in what amount and to characterize them in a spatio-temporal manner. Redox potential of a specific compart- ment or cell can be studied using a variety of techniques to identify and quantify major redox couples or redox sensitive proteins within organelles. For instance, high-performance liq- uid chromatography (HPLC) is used for the quantification of GSH/GSSG and NAD(P)H/NAD(P) + redox potentials (Jones, 2002; Takimoto et al., 2005), whereas mass spectrometry and redox Western blotting, in association with labeling of free thi- ols, are frequently used to determine the redox state of several proteins such as Trx, Trx reductase, and others (Halvey et al., 2005; Chen et al., 2006; Go et al., 2009; Go and Jones, 2013). Although these methods present high specificity for the redox couple examined and both the oxidized and reduced form can be quantified, they often require tissue/cell fractionation, dur- ing which redistribution and artifactual oxidation/reduction can occur. To overcome these problems molecular biology techniques using epitope-tagged versions of nuclear localization sequence (NLS)-Trx-1 and nuclear export sequence (NES)-Trx1 (specif- ically localized in nuclei or cytoplasm) have been developed that allow measurements without fractionation (Go et al., 2010). More recent approaches involve fluorescent imaging techniques of ROS and major redox couples within organelles in intact cells or organisms in vivo . Currently available fluorescent sen- sors for compartmentalized ROS detection can be divided into small molecule probes and genetically encoded fluorescent pro- teins. The overview of the methods presented here is by no means exhaustive and for in depth coverage the reader is referred to the following excellent reviews (Meyer and Dick, 2010; Go and Jones, 2013; Lukyanov and Belousov, 2014; Winterbourn, 2014). SMALL MOLECULE REDOX PROBES Although several small molecule fluorescent probes are available, only a few of them can be targeted to specific subcellular com- partments ( Table 1 ). It should be also mentioned that, to some extent, all these probes present limitations in terms of selectivity and sensitivity. MitoSOX Red is widely used for measurement of superoxide formation in the mitochondria of live cells (Robinson et al., 2006; Zhou et al., 2011a). MitoSOX Red indicator is a derivative of hydroethidine (HE) and contains the cationic triphenylphospho- nium substituent that is responsible for the electrophoretically driven uptake of the probe in actively respiring mitochondria. The reaction between superoxide and HE generates a highly spe- cific red fluorescent product, 2-hydroxyethidium. Nevertheless, another red fluorescent product, ethidium, can be formed from other oxidants in biological systems (Zhao et al., 2005). Thus, a simple fluorescence assay cannot distinguish between superox- ide and other oxidants. The superoxide-specific product can be detected by HPLC or mass spectrometry and only then it pro- vides a reliable method for superoxide production (Zhao et al., 2005; Zielonka et al., 2009). Reduced MitoTracker dyes, MitoTracker Orange CM- H 2 TMRos, and MitoTracker Red CM-H 2 XRos, are derivatives of dihydrotetramethyl rosamine and dihydro-X-rosamine, respec- tively. These reduced probes become fluorescent and positively charged upon their oxidation in live cells, and thus accumulate in mitochondria according to the Nernst equation (Poot et al., 1996; Kweon et al., 2001; Kaludercic et al., 2014a). As with MitoSOX Red, the quick and easy loading into the cells makes these probes very convenient. However, reduced MitoTracker dyes are not specific for single oxidant species and the fact that their Table 1 | Small molecule fluorescent redox sensitive probes and their characteristics. Redox sensor Compartment Excitation Emission Detected System used References wavelength, nm wavelength, nm ROS MitoSOX Red Mitochondria (396)510 580 O •− 2 Intact cells Robinson et al., 2006 MitoTracker Red CM-H 2 XRos Mitochondria 579 599 Not specific Intact cells Poot et al., 1996 MitoTracker Orange CM-H 2 TMRos Mitochondria 554 576 Not specific Intact cells Kweon et al., 2001 Peroxy Lucifer 1 (PL1) Cytosol 410 475/540 H 2 O 2 Intact cells Srikun et al., 2008 Nuclear Peroxy Emerald 1 (NucPE1) Nuclei 468/490 530 H 2 O 2 Intact cells, in vivo Dickinson et al., 2011 Mitochondrial Peroxy Yellow 1 (MitoPY1) Mitochondria 510 528 H 2 O 2 Intact cells Dickinson et al., 2013 SHP-Mito Mitochondria 342/383 470/545 H 2 O 2 Intact cells Masanta et al., 2012 MitoBoronic acid (MitoB) Mitochondria Mass spectroscopy H 2 O 2 In vivo Cocheme et al., 2011; Logan et al., 2014 Frontiers in Physiology | Mitochondrial Research August 2014 | Volume 5 | Article 285 | 10 Kaludercic et al. ROS compartmentalization accumulation is dependent on the mitochondrial membrane potential may lead to artifactual measurements. In order to overcome problems associated with oxidant sen- sitive dyes, new generation of fluorescent probes has been devel- oped. These are often referred to as “non-redox” probes as they contain a masked fluorophore that is released by the attack of the oxidant on the blocking group, without changing the oxidation state of the fluorophore (Winterbourn, 2014). The boronate derivatives, i.e., sensors that have boronate as block- ing group, have been synthesized for the detection of hydro- gen peroxide (H 2 O 2 ) (Miller et al., 2005). Nevertheless, it was shown that some boronate probes also respond to peroxynitrite and hypochlorous acid, thus raising some concerns regarding their specificity (Sikora et al., 2009). Peroxy Green1 (PG1) and Peroxy Crimson1 (PC1) are second-generation probes that are sensitive enough to report H 2 O 2 production at physiological signaling levels while maintaining H 2 O 2 specificity and are acti- vated by a single boronate deprotection (Miller et al., 2007). Because of their enhanced turn-on responses to H 2 O 2 , these new chemical tools are capable of detecting endogenous bursts of H 2 O 2 produced by growth factor signaling in living cells (Miller et al., 2007; Lin et al., 2013). Nevertheless, these probes were not targeted to a specific compartment. There is a wide range of compounds with different fluorophores (Dickinson et al., 2010) and adapted structures to enable targeting to mitochon- dria (Dickinson et al., 2013) and other compartments, such as nuclei and endoplasmic reticulum (ER) (Srikun et al., 2008; Dickinson et al., 2011; Woolley et al., 2012). In particular, com- bining boronate-phenol chemistry with mitochondria-targeting functional group, such as positively charged phosphonium moi- ety, led to generation of Mitochondrial Peroxy Yellow (MitoPY1), SHP-Mito, and MitoBoronic acid (MitoB) (Cocheme et al., 2012; Masanta et al., 2012; Dickinson et al., 2013). SHP-Mito is also a ratiometric probe and allows for increased penetration depth and prolonged imaging time using two-photon microscopy (Masanta et al., 2012). MitoB instead is a ratiometric mass spectrome- try probe that is rapidly converted to phenol product MitoP upon H 2 O 2 oxidation (Cocheme et al., 2011). Measurement of MitoB/MitoP ratio has been successfully used