Chen et al. reply.

Our data from intact cells demonstrate that, in addition to increasing mitoflash frequency, aldrithiol and menadione application also markedly increases basal cpYFP fluorescence intensity within the mitochondrial matrix2, 5. In addition, nanomolar concentrations of nigericin, a H+/K+ antiporter, stimulates mitoflash activity6. These responses of cpYFP in situ are unlikely to be attributable to mitochondrial alkalization. We also found that the temporal profile of mitoflash events do not always mirror the change of the mitochondrial membrane potential in cardiac and skeletal muscle cells6, 7, and this contradicts the suggestion that mitoflashes simply reflect increased proton pumping in response to membrane potential depolarization8, 9, 10. Until now, structural information about how cpYFP senses superoxide remains a mystery. In a unique class of enhanced green fluorescent protein (eGFP)-based calcium sensors, a reversible deprotonization of the chromophore occurs owing to calcium binding to a negatively charged site on the probe11. We are investigating whether a similar mechanism might underlie the reversible superoxide-sensing chemistry of cpYFP. Despite the technical issue raised by Schwarzländer et al.1, the existence of bursts of superoxide or reactive oxygen species (ROS) production in respiring mitochondria has been confirmed by several independent investigators using different probes. Two pH-insensitive, ROS probes, MitoSOX and 2′,7′-dichlorodihydrofluorescein diacetate, have validated ROS increases during cpYFP-reported ‘flashes’6, 12, 13. When used individually to avoid possible fluorescence resonance energy transfer (FRET) effects and spectral cross-contamination, these pH-insensitive ROS sensors confirmed flash events of nearly identical frequency and spatiotemporal properties as that observed for cpYFP13. Quantification of the respective contributions of superoxide and pH to mitoflash events showed that a predominant superoxide component is coincident with a modest alkalization of the mitochondrial matrix in muscle cells6. Similarly, a previous report used MitoSOX to confirm bursts of superoxide during pH alkalization events in primary astrocytes transfected with the fluorescent pH-sensor mitoSypHer14. A recent report, which is co-authored by two authors of the accompanying Comment by Schwarzländer et al.1, used roGFP2 to detect spontaneous, short-lived oxidative bursts that are accompanied by mitochondrial depolarization, transient matrix alkalization, and reversible mitochondrial ‘contractions’15, all of which we previously documented for cpYFP mitoflashes. Furthermore, in many cell types and tissues3, 5, 12, 13, 16, 17 and even in living animals2, mitoflash activity is increased by oxidants (including menadione and paraquat) and reduced by antioxidants (including mitoTEMPO and SS31). Nevertheless, given the extreme diversity and plasticity of the mitochondria proteome18, the relative contributions of superoxide and pH to cpYFP-reported mitoflash events may vary in a species-, cell-type- and context-dependent manner. It has become increasingly appreciated that mitoflash activity is a complex phenomenon, comprising multifaceted and intertwined mitochondrial processes including quantal superoxide production, membrane depolarization, membrane permeability transition, NADH depletion, matrix alkalization and swelling that masquerades as mitochondrial contraction3, 6, 14, 15, 17, 19. Ample evidence supports the notion that mitoflash activity serves as a novel and universal “frequency-coded optical readout reflecting free-radical production and energy metabolism at the single- mitochondrion level”2. The continuing debate on what drives, controls and contributes to these events does not change the fact that mitoflashes reflect a fundamental physiological phenomenon linked to energy metabolism and stress response, nor does it discount the significance of our finding that mitoflash frequency predicts lifespan at a very early age in Caenorhabditis elegans2. The Comment by Schwarzländer et al.1 focuses exclusively on the controversy of cpYFP as a superoxide sensing probe, which was originally demonstrated in several publications by Wang, Dirksen, Sheu and Cheng, and therefore these authors were invited to respond to the Comment. M.-Q. Dong and 11 authors of the original paper2 were not involved in the research that led to the discovery of cpYFP as a superoxide probe, so are not listed as authors (M.-Q. Dong was included in this Reply as a corresponding author). Download references