Respective Contribution of Mitochondrial Superoxide and pH to Mitochondria-targeted Circularly Permuted Yellow Fluorescent Protein (mt-cpYFP) Flash Activity

Wei-LaPierre, Lan; Gong, Guohua; Gerstner, Brent; Ducreux, Sylvie; Yule, David I.; Pouvreau, Stéphane; Wang, Xianhua; Sheu, Shey‐Shing; Cheng, Heping; Dirksen, Robert T.; Wang, Wang

doi:10.1074/jbc.m113.455709

Cited by 71 publications

(138 citation statements)

References 30 publications

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“…Indeed, both superoxide production and proton pumping are originated from and can be integrated by ETC electron flow. Moreover, a simultaneous loss of mitochondrial NADH signal was observed during each of the single mitochondrial flashes (52). Taken together, it is highly probable that mitochondrial flash is a composite event arising from mitochondrial respiration in vivo or in intact cells.…”

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confidence: 84%

“…This controversy could arise from the differences in in vitro calibration of cpYFP and will need to be resolved most likely with the elucidation of crystal structure of cpYFP (14,44). Nevertheless, we simultaneously monitored the cpYFP signal and a pH indicator loaded in mitochondrial matrix and found a major contribution of superoxide with a minor component of alkalization in each of the flash events (52). Considering the mixed signal in the flash event and the ongoing debate, superoxide flash has been renamed to mitoflash (45) or mitochondrial flash.…”

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confidence: 99%

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Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart

Gong

Liu

Zhang

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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tochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F 0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions. mitochondrial respiration; mitochondrial flash; electron transport chain; real-time confocal imaging; biomarker NEW & NOTEWORTHYMitochondrial flash activity is tightly coupled with the electron flow along mitochondrial electron transport chain in intact cardiac myocytes and intact heart. The positive correlation between flash frequency and oxygen consumption rate highly suggests that flash frequency could be used as a novel biomarker for mitochondrial respiration in vivo.

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confidence: 84%

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confidence: 99%

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart

Gong

Liu

Zhang

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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“…We and others reported cyclosporine A-sensitive large conductance channel activities ( > 1 nS single channel conductance) in the IMM (154). This channel also has transient and subconductance ( < 300 pS) states, and these channel properties contribute to an important physiological or pathological role by preventing [Ca 2 + ] mt overload working as a Ca 2 + efflux mechanism (66) and/or by regulating mitochondrial ROS generation as described as an mSOF (188,190). On mPTP opening, the IMM no longer maintains a barrier to protons, which leads to depolarization of DJ m , followed by the inhibition of ATP production.…”

Section: Mitochondrial K 1 Channels and Mitochondrial Redox Signalingmentioning

confidence: 99%

“…- (190). Future studies will also be directed toward inventing a novel O 2 --sensitive biomarker to reduce its pH sensitivity.…”

Section: Fig 5 Ros-dependent Regulation Of Cellular Ca 21 Handing Amentioning

confidence: 99%

Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

O‐Uchi

Ryu

Jhun

et al. 2014

Antioxidants & Redox Signaling

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Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca 2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K + channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987-1006.

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“…Mitoflash has been detected by multiple indicators including mt-cpYFP, which dually senses superoxide and pH (Wei-LaPierre et al, 2013), mitoSOX for superoxide and 2,7-dichlorodihydrofluorescein diacetate (DCF) for total ROS , grx1-roGFP2 for redox potential (Breckwoldt et al, 2014;Wang et al, 2016), mitoSypHer, pHTomato and SNARF-1 for matrix pH (SantoDomingo et al, 2013;Wang et al, 2016;Wei-LaPierre et al, 2013), and TMRM or TMRE for mitochondrial membrane potential (Wang et al, 2008;Wei et al, 2011). In addition, label-free imaging of NADH and FAD autofluorescence also reveals a transient oxidation of NADH and FADH 2 Wang et al, 2016;Wei-LaPierre et al, 2013). Thus, individual mitoflashes consist of multiple signals including bursting superoxide production, transient matrix alkalization, oxidative redox shift and mitochondrial membrane potential depolarization Wang et al, 2008Wang et al, , 2016, reflecting electrical and chemical excitation of the organelle.…”

Section: Introductionmentioning

confidence: 99%

Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes

Jian

Hou

et al. 2017

Journal of Cell Science

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Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis.

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Respective Contribution of Mitochondrial Superoxide and pH to Mitochondria-targeted Circularly Permuted Yellow Fluorescent Protein (mt-cpYFP) Flash Activity

Cited by 71 publications

References 30 publications

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart

Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes

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