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.

A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential

Abstract

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer’s and Parkinson’s diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Design of the MAL probe.
Fig. 2: In vitro validation of the MAL3 probe for imaging and monitoring of ΔΨm.
Fig. 3: In vivo validation of the MAL3 probe for imaging and monitoring of ΔΨm.
Fig. 4: Application of the MAL3 probe for monitoring ΔΨm in young and old mice and investigation of the effect of NR-enriched diet on ΔΨm in old mice.
Fig. 5: Application of MAL3 for monitoring ΔΨm in BAT and tumor-bearing mice in vivo.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. CCDC nos. 1940412, 1940411, 1940410 and 1940409 for compounds 7, 9, 13 and 16, contain the supplementary crystallographic data for this paper. These data can be obtained, free of charge, from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.

References

  1. 1.

    Herst, P. M., Rowe, M. R., Carson, G. M. & Berridge, M. V. Functional mitochondria in health and disease. Front. Endocrinol. (Lausanne) 8, 296 (2017).

    Google Scholar 

  2. 2.

    Duchen, M. R. Mitochondria in health and disease: perspectives on a new mitochondrial biology. Mol. Aspects Med. 25, 365–451 (2004).

    CAS  PubMed  Google Scholar 

  3. 3.

    Wang, Y., Xu, E., Musich, P. R. & Lin, F. Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure. CNS Neurosci. Ther. 25, 816–824 (2019).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Zorova, L. D. et al. Mitochondrial membrane potential. Anal. Biochem. 552, 50–59 (2018).

    CAS  PubMed  Google Scholar 

  5. 5.

    Momcilovic, M. et al. In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer. Nature 575, 380–384 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Lee, J. H. et al. The role of adipose tissue mitochondria: regulation of mitochondrial function for the treatment of metabolic diseases. Int. J. Mol. Sci. 20, 4924 (2019).

    CAS  PubMed Central  Google Scholar 

  7. 7.

    Vyas, S., Zaganjor, E. & Haigis, M. C. Mitochondria and cancer. Cell 166, 555–566 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Weinberg, S. E. & Chandel, N. S. Targeting mitochondria metabolism for cancer therapy. Nat. Chem. Biol. 11, 9–15 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Perry, S. W., Norman, J. P., Barbieri, J., Brown, E. B. & Gelbard, H. A. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques 50, 98–115 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Rottenberg, H. & Wu, S. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta 1404, 393–404 (1998).

    CAS  PubMed  Google Scholar 

  11. 11.

    Gerencser, A. A. et al. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. J. Physiol. 590, 2845–2871 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Madar, I. et al. Characterization of uptake of the new PET imaging compound F-18-fluorobenzyl triphenyl phosphonium in dog myocardium. J. Nucl. Med. 47, 1359–1366 (2006).

    CAS  Google Scholar 

  13. 13.

    Kim, D. Y. et al. Evaluation of a mitochondrial voltage sensor, (18F-fluoropentyl)triphenylphosphonium cation, in a rat myocardial infarction model. J. Nucl. Med. 53, 1779–1785 (2012).

    CAS  PubMed  Google Scholar 

  14. 14.

    Logan, A. et al. Assessing the mitochondrial membrane potential in cells and in vivo using targeted click chemistry and mass spectrometry. Cell Metab. 23, 379–385 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Gaskova, D., DeCorby, A. & Lemire, B. D. DiS-C-3(3) monitoring of in vivo mitochondrial membrane potential in C. elegans. Biochem. Biophys. Res. Commun. 354, 814–819 (2007).

    CAS  PubMed  Google Scholar 

  16. 16.

    Sasagawa, S. et al. In vivo detection of mitochondrial dysfunction induced by clinical drugs and disease-associated genes using a novel dye ZMJ214 in zebrafish. ACS Chem. Biol. 11, 381–388 (2016).

    CAS  PubMed  Google Scholar 

  17. 17.

    Massoud, T. F. & Gambhir, S. S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Gene Dev. 17, 545–580 (2003).

    CAS  PubMed  Google Scholar 

  18. 18.

    Sweeney, T. J. et al. Visualizing the kinetics of tumor-cell clearance in living animals. Proc. Natl Acad. Sci. USA 96, 12044–12049 (1999).

    CAS  PubMed  Google Scholar 

  19. 19.

    Mezzanotte, L., van ‘t Root, M., Karatas, H., Goun, E. A. & Lowik, C. In vivo molecular bioluminescence imaging: new tools and applications. Trends Biotechnol. 35, 640–652 (2017).

    CAS  PubMed  Google Scholar 

  20. 20.

    Rathbun, C. M. & Prescher, J. A. Bioluminescent probes for imaging biology beyond the culture dish. Biochemistry 56, 5178–5184 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Su, T. A., Bruemmer, K. J. & Chang, C. J. Caged luciferins for bioluminescent activity-based sensing. Curr. Opin. Biotechnol. 60, 198–204 (2019).

    CAS  PubMed  Google Scholar 

  22. 22.

    Maric, T. et al. Bioluminescent-based imaging and quantification of glucose uptake in vivo. Nat. Methods 16, 526–532 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Zagozdzon, A. M. et al. Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice. BMC Cancer 12, 209 (2012).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Cao, Y. A. et al. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc. Natl Acad. Sci. USA 101, 221–226 (2004).

    CAS  PubMed  Google Scholar 

  25. 25.

    Manni, I., de Latouliere, L., Gurtner, A. & Piaggio, G. Transgenic animal models to visualize cancer-related cellular processes by bioluminescence imaging. Front. Pharmacol. 10, 235 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Saxon, E. & Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000).

    CAS  PubMed  Google Scholar 

  27. 27.

    Myers, E. L. & Raines, R. T. A phosphine-mediated conversion of azides into diazo compounds. Angew. Chem. Int. Ed. 48, 2359–2363 (2009).

    CAS  Google Scholar 

  28. 28.

    Sundhoro, M., Jeon, S., Park, J., Ramstrom, O. & Yan, M. Perfluoroaryl azide Staudinger reaction: a fast and bioorthogonal reaction. Angew. Chem. Int. Ed. 56, 12117–12121 (2017).

    CAS  Google Scholar 

  29. 29.

    Cohen, A. S., Dubikovskaya, E. A., Rush, J. S. & Bertozzi, C. R. Real-time bioluminescence imaging of glycans on live cells. J. Am. Chem. Soc. 132, 8563–8565 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Murphy, M. P. Targeting lipophilic cations to mitochondria. Biochim. Biophys. Acta 1777, 1028–1031 (2008).

    CAS  PubMed  Google Scholar 

  31. 31.

    Prime, T. A. et al. A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury. Proc. Natl Acad. Sci. USA 106, 10764–10769 (2009).

    CAS  PubMed  Google Scholar 

  32. 32.

    Jameson, V. J. A. et al. Synthesis of triphenylphosphonium vitamin E derivatives as mitochondria-targeted antioxidants. Tetrahedron 71, 8444–8453 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Ross, M. F. et al. Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology. Biochemistry (Mosc.) 70, 222–230 (2005).

    CAS  PubMed  Google Scholar 

  34. 34.

    Woodroofe, C. C. et al. N-alkylated 6′-aminoluciferins are bioluminescent substrates for Ultra-Glo and QuantiLum luciferase: new potential scaffolds for bioluminescent assays. Biochemistry 47, 10383–10393 (2008).

    CAS  PubMed  Google Scholar 

  35. 35.

    Zhang, J. H., Chung, T. D. Y. & Oldenburg, K. R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    CAS  PubMed  Google Scholar 

  36. 36.

    Nicholls, D. G. Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons. J. Biol. Chem. 281, 14864–14874 (2006).

    CAS  PubMed  Google Scholar 

  37. 37.

    Erez, Y. et al. Comparative study of the photoprotolytic reactions of D-luciferin and oxyluciferin. J. Phys. Chem. A 116, 7452–7461 (2012).

    CAS  PubMed  Google Scholar 

  38. 38.

    Wyatt, C. N. & Buckler, K. J. The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells. J. Physiol. 556, 175–191 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Lou, P. H. et al. Mitochondrial uncouplers with an extraordinary dynamic range. Biochem. J. 407, 129–140 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Manago, A. et al. Early effects of the antineoplastic agent salinomycin on mitochondrial function. Cell Death Dis. 6, e1930 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Martens, C. R. et al. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults. Nat. Commun. 9, 1286 (2018).

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Mouchiroud, L. et al. The NAD+/Sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling. Cell 154, 430–441 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Vannini, N. et al. The NAD-booster nicotinamide riboside potently stimulates hematopoiesis through increased mitochondrial clearance. Cell Stem Cell 24, 405–418 (2019).

    CAS  PubMed  Google Scholar 

  44. 44.

    Lowell, B. B. & Spiegelman, B. M. Towards a molecular understanding of adaptive thermogenesis. Nature 404, 652–660 (2000).

    CAS  PubMed  Google Scholar 

  45. 45.

    Cypess, A. M. et al. Activation of human brown adipose tissue by a β3-adrenergic receptor agonist. Cell Metab. 21, 33–38 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Virtanen, K. A. Activation of human brown adipose tissue (BAT): focus on nutrition and eating. Handb. Exp. Pharmacol 251, 349–357 (2018).

    Google Scholar 

  47. 47.

    Yang, Y. et al. Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy. J. Cell. Physiol. 231, 2570–2581 (2016).

    CAS  PubMed  Google Scholar 

  48. 48.

    Schondorf, D. C. et al. The NAD+ precursor nicotinamide riboside rescues mitochondrial defects and neuronal loss in iPSC and fly models of Parkinson’s disease. Cell Rep. 23, 2976–2988 (2018).

    PubMed  Google Scholar 

  49. 49.

    Szabadkai, G. & Duchen, M. R. Mitochondria mediated cell death in diabetes. Apoptosis 14, 1405–1423 (2009).

    CAS  PubMed  Google Scholar 

  50. 50.

    Kleinert, M. et al. Animal models of obesity and diabetes mellitus. Nat. Rev. Endocrinol. 14, 140–162 (2018).

    PubMed  Google Scholar 

  51. 51.

    Zha, L. et al. The histone demethylase UTX promotes brown adipocyte thermogenic program via coordinated regulation of H3K27 demethylation and acetylation. J. Biol. Chem. 290, 25151–25163 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Erlich-Hadad, T. et al. TAT-MTS-MCM fusion proteins reduce MMA levels and improve mitochondrial activity and liver function in MCM-deficient cells. J. Cell. Mol. Med. 22, 1601–1613 (2018).

    CAS  PubMed  Google Scholar 

  53. 53.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the EPFL NMR facility staff and especially P. Mieville for help with acquiring 2D spectra, the EPFL XRD facility staff for help with crystal structure determination, and the EPFL MS facility for help with HRMS measurements. We thank O. Naveiras and A. Oggier (ISREC, EPFL), who kindly provided NR-enriched and control diets. We also thank G. Karateev for help and advice regarding the synthesis of TPP-CL2. We appreciate the help of R. Combe and M. Kulagin, members of the CPG facility at EPFL, in measuring the oxygen consumption by mice. We thank the Swiss National Foundation (grant no. 31003A_150134) for funding this work.

Author information

Affiliations

Authors

Contributions

E.A.G. conceptualized the study and acquired the funding. E.A.G. and A.A.B. designed the experiments. A.A.B., R.S. and N.S. synthesized the compounds. A.A.B. performed the experiments and analyzed the data. A.A.B., A.H. and U.D.M. designed and performed cellular OCR measurements. T.M. measured the clearance of TPP-CL2 in vivo, and designed and drew the graphical abstract. G.B. assisted in measurement of the nigericin effect in cells with TMRM. E.A.G. and A.A.B. wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Elena A. Goun.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–28, Tables 1–3 and notes 1–3.

Reporting Summary

Supplementary Video 1

Working principle of MAL probe.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bazhin, A.A., Sinisi, R., De Marchi, U. et al. A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential. Nat Chem Biol 16, 1385–1393 (2020). https://doi.org/10.1038/s41589-020-0602-1

Download citation

Further reading

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