The JGP series Perspectives on: SGP Symposium on Mitochondrial Physiology and Medicine includes articles by Wei and Dirksen (2012) and by Santo-Domingo and Demaurex (2012), which highlight two fields of research in mitochondrial physiology experiencing renewed interest thanks to the development of new genetically encoded probes: mt-cpYFP (a mitochondria-targeted superoxide sensor) and mitoSypHer (a mitochondria-targeted pH probe). Interestingly, both articles discuss the detection of spontaneous spatiotemporal restricted elevations in the fluorescence intensity of the sensors (“flashes”). In one case, flashes are attributed to a quantal production of superoxide (O2) (Wei and Dirksen, 2012) and in the other one to spontaneous elevations in mitochondrial pH (Santo-Domingo and Demaurex, 2012). In the following paragraphs, we discuss the possible nature of the flashes in the light of the properties of each sensor.

Discovery and generation of the mt-cpYFP and mitoSypHer

It is amusing to consider that both probes have been serendipitously discovered while running experiments on “old sensors.” Mt-cpYFP is derived from the mitochondria-targeted calcium (Ca2+) indicator ratiometric pericam, described by Nagai et al. in 2001. It was used as the core of this Ca2+ probe before its sensitivity for O2 was proposed by Wang et al. in 2008. cpYFP was generated by circular permutation and point mutation of a YFP variant, EYFP(V68L/Q69K), with the two original termini being connected by the linker VDGGSGGTG (Nagai et al., 2001).

SypHer is a mutated form of Hyper, a genetically encoded sensor for hydrogen peroxide, developed by Belousov et al. in 2006. Hyper is composed of a cpYFP inserted into Oxy-RD, the regulatory domain of Escherichia coli OxyR, which is specifically sensitive to H2O2. cpYFP was obtained by circular permutation of the EYFP sequence from the plasmid pEYFP-N1 (Takara Bio Inc.). The linker VDGGSGGTG was also used between the two termini. Several mutations were introduced to optimize folding and chromophore maturation. SypHer was developed by Poburko et al. (2011) by mutating one of the two H2O2-sensing cysteine residues of the OxyRD domain of Hyper (C199S), following Belousov et al. (2006). This single mutation rendered SypHer unresponsive to H2O2 while preserving its pH sensitivity.

cpYFP: A pH or superoxide sensor?

MitoSypHer as a superoxide sensor?

Even if the two cpYFPs constituting the cores of mt-cpYFP and mitoSypHer have been engineered by two different groups, they only differ by seven residues. Strikingly, none of these residues should be involved in O2 sensing. According to Wang et al. (2008), two cysteines are involved in mt-cpYFP O2 sensing: C171 and C193. These two residues are present in the cpYFP contained in the Hyper/SypHer. Surprisingly, although the O2 sensitivity of mt-cpYFP has been repeatedly challenged, the reciprocal hypothesis that Hyper/SypHer might also detect O2 has been overlooked. The only article (Belousov et al., 2006) investigating the sensitivity of Hyper to O2 shows no increase in its fluorescence upon the addition of 30 µM xanthine and 25 mU xanthine oxidase in vitro. However, the in vitro characterizations of Hyper and mt-cpYFP by Belousov et al. (2006) and Wang et al. (2008), respectively, have been conducted under different experimental conditions. Hyper isolation procedures were performed in a buffer containing 5 mM 2-mercaptoethanol, and characterization of Hyper specificity was performed in a buffer containing 0.5 mM 2-mercaptoethanol. The calibration solution of Wang et al. (2008) contained HEPES, KCl, and EDTA, but the 10 mM of reduced dithiothreitol used in previous steps was removed from the calibration solution. It has been shown that the presence of oxidants or of 2-mercaptoethanol in the recording solution affects the photobehavior of diverse fluorescent proteins (Bogdanov et al., 2009). The proposed mechanism involves the formation of radical intermediates of the chromophore. It is thus possible that, in the study of Belousov et al. (2006), the 2-mercaptoethanol present in the calibration medium affects the reactivity of the fluorescent protein toward O2. Interestingly, Bogdanov et al. (2009) reported variable photobehaviors of the GFP probes among different cells lines and in a same line among different cellular compartments. They attributed this phenomenon to differences in redox environment.

By transfecting mouse skeletal muscle fibers with either mitoHyper or its C199 mutated version (mitoSypHer), we were able to detect transient increases in fluorescence (Fig. 1), which resemble the superoxide flashes that we already described in skeletal muscle (Pouvreau, 2010). Like mt-cpYFP flashes, mitoHyper/SypHer transients are accompanied by a depolarization of the mitochondria. Moreover, the mitoHyper/SypHer transients are simultaneous with increases in fluorescence intensity of MitoSOX, a mitochondrial targeted synthetic probe for O2. These results are in accordance with Azarias and Chatton (2011), who reported flashes in mitoSypHer fluorescence accompanied by increases in MitoSOX fluorescence in astrocytes. However, because mitoSypHer was initially described as a pH sensor, Azarias and Chatton interpreted the spontaneous fluorescence flashes as pH transients. MitoSOX sensitivity to O2 is well established, even if its specificity for O2 over other reactive oxygen species (ROS) has been questioned. This limitation of the sensor is of minor importance here, as cpYFP emission is unchanged upon the addition of various ROS (Wang et al., 2008). The least we can conclude from this set of experiments is that an increase in ROS production is simultaneous to the flashes. Thus, in light of these data, further work appears necessary to reevaluate the potential superoxide sensitivity of mitoSypHer.

Mt-cpYFP as a pH sensor?

In a letter to the editor in the journal Free Radical Biology and Medicine, published a few months after the seminal article of Wang et al. (2008), Muller questioned the validity of mt-cpYFP as a superoxide probe, mostly on the basis of the peculiar pharmacology of the flashes (Muller, 2009), and proposed that mt-cpYFP detects ATP. The different points of this letter have been answered (Huang et al., 2011), and the hypothesis of the sensitivity of mt-cpYFP to ATP was discarded. We do believe nonetheless that the main question raised by Muller (2009), the unexpected effect of complex III inhibitor antimycin A on superoxide flash frequency, requires further investigations. Such a work might also provide new insights into the mechanisms underlying ROS production by the electron transport chain.

A second critical evaluation of the superoxide nature of the flashes was published by Schwarzländer et al. (2011). In their article, the authors claimed that the mt-cpYFP flashes are not superoxide flashes but pH flashes. The authors based their conclusion on (a) the absence of changes in global fluorescence intensity of mt-cpYFP upon manipulation of mitochondria O2 levels in their experimental model (isolated mitochondria preparation from the plant Arabidopsis), (b) the sensitivity of cpYFP to pH, and (c) the fact that manipulating mitochondrial pH affects the properties of superoxide flashes. With cpYFP being, just like several fluorescent proteins, sensitive to pH under physiological conditions (Nagai et al., 2001; Belousov et al., 2006; Wang et al., 2008), the real matter of discussion is whether mt-cpYFP is also detecting O2. We and others demonstrated in mammalian cells that changes in the O2 content affect the properties of the flashes: the addition of antioxidants decreases the frequency of flashes (Wang et al., 2008; Pouvreau, 2010; Huang et al., 2011), whereas menadione, which mediates O2 release in mitochondria, and knocking down superoxide dismutase 2 increase flash frequency (Huang et al., 2011). Furthermore, Tiron treatment increased significantly the time to peak of mt-cpYFP flashes, which would be expected if flashes reflected transient increases in O2 (Pouvreau, 2010). Results relying on manipulation of mitochondrial pH should be carefully interpreted, as O2 production has been shown to be dependent on the pH gradient across the mitochondrial inner membrane (Lambert and Brand, 2004). Interestingly, no pH flashes have been reported by several groups studying mitochondrial pH using other probes: mt-EYFP (pKa = 7; see, for instance, Wang et al., 2008) and mitoAlpHi (pKa = 8.5; see, for instance, Abad et al., 2004). Importantly, mt-cpYFP pKa is similar to that of mitoAlpHi (Nagai et al., 2001). Given the sensitivity of cpYFP to pH, it is possible that at least part of the increase in the mt-cpYFP fluorescence during a flash is caused by detection of changes in pH. However, further experiments are required to validate this hypothesis. Measurement of flashes simultaneously with cpYFP and a red-shifted chemically distinct pH-sensitive probe would be particularly informative.

Are we all looking at the same events?

Mt-cpYFP and mitoSypHer are both ratiometric probes (excitation wavelengths being 405/488 or 491 nm in the studies with mt-cpYFP, and 420/490 nm with mito-SypHer), and their pKa values are comparable: ≈8.6 for mt-cpYFP (Schwarzländer et al., 2011; Wei and Dirksen, 2012) and 8.71 for mito-SypHer (Poburko et al., 2011). Unfortunately, currently available data do not allow a fine comparison of the respective spectral behavior of the two probes, nor of their spectral response to pH versus O2, as the spectra provided by the different groups have been acquired under different conditions (Belousov et al., 2006; Wang et al., 2008; Poburko et al., 2011; Schwarzländer et al., 2011). Data showing spectral properties of mt-cpYFP and mitoSypHer, as well as spectral response of the two probes to pH and O2 under the same conditions, would be paramount.

Based on their 2011 study stating that mt-cpYFP was in fact a pH sensor, Schwarzländer et al. (2012) published an article looking at spontaneous membrane depolarizations (pulsing) in mitochondria isolated from Arabidopsis, which they claim are accompanied by pH transients mirroring the kinetics of the depolarization transients, as well as mitochondrial Ca2+ transients. The authors suggest that the pH flash is a compensatory mechanism to depolarization. Interestingly, pulsing is affected by changes in ROS level. The transient events reported in this study differ from the ones reported in mammalian cells using mt-cpYFP (Wang et al., 2008, Pouvreau, 2010) or mitoSypHer (Azarias and Chatton, 2011) in several aspects. First, Wang et al. (2008), Pouvreau (2010), and Azarias and Chatton (2011) showed no change or a decrease in mitochondrial Ca2+ level during cpYFP transients. This discrepancy might be caused by the difference in experimental models (mammalian cells vs. isolated mitochondria from plants). Second, membrane depolarizations reported by Wang et al. (2008) and Pouvreau (2010) do not systematically mirror the mt-cpYFP flashes: they can be square-shaped or long-lasting, or in other words, of longer duration than the flashes. Here, we also report a long-lasting depolarization accompanying a mitoHyper flash (Fig. 1 A). Furthermore, Pouvreau (2010) showed that one third of the spontaneous mitochondrial depolarizations are devoid of flashes. Hence, the hypothesis that cpYFP flashes are alkalinization events that preserve the proton-motive force during spontaneous mitochondria depolarizations seems to be questionable in our system. However, in keeping with the chemiosmotic theory, variations of pH gradient are expected during drops in mitochondrial membrane potential. Hence, pH variation during mitochondrial depolarization should be investigated using different pH probes.

Unfortunately, most of the data collected with mt-cpYFP or mitoHyper/SypHer have been acquired by different groups in different systems. To our knowledge, the results reported here are the first performed with these probes under the same conditions. Our data suggest that the two probes are detecting the same events, and that these events are concomitant with MitoSOX steps. Needless to say, this constitutes only a small step in regard to the work required to answer the questions: Are mt-cpYFP and mitoSypHer flashes reporting the same events? Are the events reflecting changes in pH or in O2 level? Further tests conducted by different groups in their different systems are necessary.

Conclusion

Although the current available literature provides convincing evidences that cpYFP acts as a superoxide sensor, the hypothesis that the reported “superoxide flashes” are at least partially caused by pH detection cannot be refuted. Indeed, the pH sensitivity of cpYFP, as well as of many other GFP-based sensors, is well established. Further studies are required to clarify the remaining concerns on the superoxide nature of the flashes. Unraveling the chemical nature and mechanisms of superoxide sensing by cpYFP should definitely resolve the disagreement. Beyond this controversy, the discovery of mitoSypHer/mt-cpYFP fluorescence transients provides exciting insights into the temporal and spatial functioning of mitochondria. The triggering of mitoSypHer flashes by localized decreases in ATP concentration has been shown (Azarias and Chatton, 2011), and could explain the well-defined localization of flashes among a background of quiescent mitochondria. MitoSypHer and mt-cpYFP flashes are accompanied by transient depolarization, whose mechanism is not unraveled yet. Although an opening of the mitochondrial permeability transition pore can explain this depolarization in cardiac myocytes (Wang et al., 2008), such a mechanism was not observed in skeletal muscle (Pouvreau, 2010). All these exciting questions will undoubtedly be the subject of future research studies.

Acknowledgments

We thank Prof. Lukyanov (Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) for kindly providing the C199 construct.

This work was supported by grants from the Université Lyon 1 and the Centre National de la Recherche Scientifique.

Edward N. Pugh Jr. served as editor.

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