Mitochondria-targeted cpYFP: pH or superoxide sensor?

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 mito-chondria-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 (O 2 ) (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 mito-chondria-targeted calcium (Ca 2+) indicator ratiometric pericam, described by Nagai et al. in 2001. It was used as the core of this Ca 2+ probe before its sensitivity for O 2  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 Esche-richia coli OxyR, which is specifically sensitive to H 2 O 2. 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 H 2 O 2-sensing cysteine residues of the OxyRD domain of Hyper (C199S), following Belousov et al. (2006). This single mutation rendered SypHer unresponsive to H 2 O 2 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 …