Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cheng et al. reply

Abstract

Replying to M. Schwarzländer et al. Nature 514, 10.1038/nature13858 (2014)

In the accompanying Comment1, Schwarzländer et al. challenged our recent study2 because they failed to reproduce our previous finding that the fluorescence intensity of purified circularly permuted yellow fluorescent protein (cpYFP) increases in response to oxygen and superoxide anions produced by xanthine (X) plus xanthine oxidase (XO)3. Starting from a ‘fully reduced’ state (incubation with 10 mM dithiothreitol for >3 h) and in the presence of 75 mM HEPES, we demonstrated that cpYFP exhibits a twofold fluorescence increase after oxygenation, and an additional twofold increase after the subsequent addition of X plus XO, which could not be accounted for by solvent (potassium hydroxide)-induced alkalization. Furthermore, the xanthine plus xanthine oxidase-induced increase in cpYFP fluorescence was reversed by Cu/Zn superoxide dismutase (600 U ml−1). We also found that the fluorescence intensity of fully reduced cpYFP increased >fourfold after incubation with 1 mM aldrithiol. Notably, recombinant cpYFP purified in the absence of dithiothreitol treatment exhibits a high fluorescence comparable to that of the fully oxidized state, indicating the high susceptibility of cpYFP to oxidation in non-reducing environments3. Therefore, ensuring a fully reduced state of cpYFP is essential for the probe to sense superoxide in vitro. This property is probably the reason that the probe functions readily as a reversible superoxide biosensor when targeted to the reduced environment of the mitochondrial matrix. Unfortunately, from the brief description of the methods and limited data provided by Schwarzländer et al.1, it is not possible to determine whether cpYFP was fully reduced in their experiments, or whether sufficient precautions were taken to prevent oxidation of the probe. Moreover, in our experiments cpYFP was expressed in Escherichia coli BL21(DE3)LysS cells, whereas Schwarzländer et al.1 used E. coli Origami, a trxB (thioredoxin reductase) mutant strain that also lacks glutathione reductase needed to fully limit cysteine oxidation4, which could result in an increased oxidative status of their purified cpYFP rendering it non-responsive to superoxide.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

References

  1. Schwarzländer, M. et al. The ‘mitoflash’ probe cpYFP does not respond to superoxide. Nature 514, http://dx.doi.org/10.1038/nature13858 (2014)

    Article  Google Scholar 

  2. Shen, E.-Z. et al. Mitoflash frequency in early adulthood predicts lifespan in Caenorhabditis elegans. Nature 508, 128–132 (2014)

    ADS  CAS  Article  Google Scholar 

  3. Wang, W. et al. Superoxide flashes in single mitochondria. Cell 134, 279–290 (2008)

    CAS  Article  Google Scholar 

  4. Derman, A. I., Prinz, W. A., Belin, D. & Beckwith, J. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science 262, 1744–1747 (1993)

    ADS  CAS  Article  Google Scholar 

  5. Huang, Z. et al. Response to “A critical evaluation of cpYFP as a probe for superoxide”. Free Radic. Biol. Med. 51, 1937–1940 (2011)

    CAS  Article  Google Scholar 

  6. Wei-LaPierre, L. et al. Respective contribution of mitochondrial superoxide and pH to mitochondria-targeted circularly permuted yellow fluorescent protein (mt-cpYFP) flash activity. J. Biol. Chem. 288, 10567–10577 (2013)

    CAS  Article  Google Scholar 

  7. Li, K. et al. Superoxide flashes reveal novel properties of mitochondrial reactive oxygen species excitability in cardiomyocytes. Biophys. J. 102, 1011–1021 (2012)

    ADS  CAS  Article  Google Scholar 

  8. Schwarzländer, M. et al. Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory bioenergetics in Arabidopsis. Plant Cell 24, 1188–1201 (2012)

    Article  Google Scholar 

  9. Schwarzlander, M. et al. Mitochondrial ‘flashes’: a radical concept repHined. Trends Cell Biol. 22, 503–508 (2012)

    Article  Google Scholar 

  10. Santo-Domingo, J., Giacomello, M., Poburko, D., Scorrano, L. & Demaurex, N. OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange. EMBO J. 32, 1927–1940 (2013)

    CAS  Article  Google Scholar 

  11. Tang, S. et al. Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments. Proc. Natl Acad. Sci. USA 108, 16265–16270 (2011)

    ADS  CAS  Article  Google Scholar 

  12. Pouvreau, S. Superoxide flashes in mouse skeletal muscle are produced by discrete arrays of active mitochondria operating coherently. PLoS ONE 5, e13035 (2010)

    ADS  Article  Google Scholar 

  13. Zhang, X. et al. Superoxide constitutes a major signal of mitochondrial superoxide flash. Life Sci. 93, 178–186 (2013)

    CAS  Article  Google Scholar 

  14. Azarias, G. & Chatton, J. Y. Selective ion changes during spontaneous mitochondrial transients in intact astrocytes. PLoS ONE 6, e28505 (2011)

    ADS  CAS  Article  Google Scholar 

  15. Breckwoldt, M. O. et al. Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo. Nature Med. 20, 555–560 (2014)

    CAS  Article  Google Scholar 

  16. Hou, T. et al. Synergistic triggering of superoxide flashes by mitochondrial Ca2+ uniport and basal reactive oxygen species elevation. J. Biol. Chem. 288, 4602–4612 (2013)

    CAS  Article  Google Scholar 

  17. Ma, Q. et al. Superoxide flashes: early mitochondrial signals for oxidative stress-induced apoptosis. J. Biol. Chem. 286, 27573–27581 (2011)

    CAS  Article  Google Scholar 

  18. Pagliarini, D. J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008)

    CAS  Article  Google Scholar 

  19. Hou, T., Wang, X., Ma, Q. & Cheng, H. Mitochondrial flashes: new insights into mitochondrial ROS signaling and beyond. J. Physiol. (Lond.) 592, 3703–3713 (2014)

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Heping Cheng or Meng-Qiu Dong.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cheng, H., Wang, W., Wang, X. et al. Cheng et al. reply. Nature 514, E14–E15 (2014). https://doi.org/10.1038/nature13859

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13859

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing