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.

Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques

Abstract

Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. Long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein–cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. These procedures can easily be modified and adapted to different cell types. Cell culture and preparation of the samples are estimated to take 1 d for methods (i) and (ii), and 3 d for method (iii). The entire experiment, including analyses, takes 2 h.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Representative images and kinetics of HeLa cells stained using the Co2+–calcein technique.
Figure 2: Representative images and kinetics of HeLa cells stained with TMRM (Fire LUT was applied).
Figure 3: Representative images and kinetics of HeLa cells expressing mitochondrially targeted GFP.

References

  1. Zoratti, M. & Szabo, I. The mitochondrial permeability transition. Biochim. Biophys. Acta 1241, 139–176 (1995).

    Article  PubMed  Google Scholar 

  2. Kwong, J.Q. & Molkentin, J.D. Physiological and pathological roles of the mitochondrial permeability transition pore in the heart. Cell Metab. 21, 206–214 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bernardi, P. & Di Lisa, F. The mitochondrial permeability transition pore: molecular nature and role as a target in cardioprotection. J. Mol. Cell. Cardiol. 78, 100–106 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bonora, M. et al. Molecular mechanisms of cell death: central implication of ATP synthase in mitochondrial permeability transition. Oncogene 34, 1475–1486 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Halestrap, A.P. The C ring of the F1Fo ATP synthase forms the mitochondrial permeability transition pore: a critical appraisal. Front. Oncol. 4, 234 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Halestrap, A.P. & Richardson, A.P. The mitochondrial permeability transition: a current perspective on its identity and role in ischaemia/reperfusion injury. J. Mol. Cell. Cardiol. 78, 129–141 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Morciano, G. et al. Molecular identity of the mitochondrial permeability transition pore and its role in ischemia-reperfusion injury. J. Mol. Cell. Cardiol. 78, 142–153 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Bonora, M. et al. Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition. Cell Cycle 12, 674–683 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. De Marchi, E., Bonora, M., Giorgi, C. & Pinton, P. The mitochondrial permeability transition pore is a dispensable element for mitochondrial calcium efflux. Cell Calcium 56, 1–13 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Alavian, K.N. et al. An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore. Proc. Natl. Acad. Sci. USA 111, 10580–10585 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Azarashvili, T. et al. Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening. Cell Calcium 55, 69–77 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Crofts, A.R. & Chappell, J.B. Calcium ion accumulation and volume changes of isolated liver mitochondria. Reversal of calcium ion-induced swelling. Biochem. J. 95, 387–392 (1965).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chappell, J.B. & Crofts, A.R. Calcium ion accumulation and volume changes of isolated liver mitochondria. Calcium ion-induced swelling. Biochem. J. 95, 378–386 (1965).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hausenloy, D.J., Duchen, M.R. & Yellon, D.M. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovas. Res. 60, 617–625 (2003).

    Article  CAS  Google Scholar 

  15. Wieckowski, M.R. & Wojtczak, L. Fatty acid-induced uncoupling of oxidative phosphorylation is partly due to opening of the mitochondrial permeability transition pore. FEBS Lett. 423, 339–342 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Gautier, C.A. et al. Regulation of mitochondrial permeability transition pore by PINK1. Mol. Neurodegener. 7, 22 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Crompton, M. The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341, 233–249 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wong, R., Steenbergen, C. & Murphy, E. Mitochondrial permeability transition pore and calcium handling. Methods Mol. Biol. 810, 235–242 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Marcu, R., Neeley, C.K., Karamanlidis, G. & Hawkins, B.J. Multi-parameter measurement of the permeability transition pore opening in isolated mouse heart mitochondria. J. Vis. Exp. 67 (2012).

  20. Varanyuwatana, P. & Halestrap, A.P. The roles of phosphate and the phosphate carrier in the mitochondrial permeability transition pore. Mitochondrion 12, 120–125 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Piwocka, K. et al. A novel apoptosis-like pathway, independent of mitochondria and caspases, induced by curcumin in human lymphoblastoid T (Jurkat) cells. Exp. Cell Res. 249, 299–307 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Griffiths, E.J. & Halestrap, A.P. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem. J. 307, 93–98 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gillessen, T., Grasshoff, C. & Szinicz, L. Biomed. Pharmacother. 56, 186–193 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Hausenloy, D., Wynne, A., Duchen, M. & Yellon, D. Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection. Circulation 109, 1714–1717 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Petronilli, V. et al. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys. J. 76, 725–734 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Woollacott, A.J. & Simpson, P.B. High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells. J. Biomol. Screen. 6, 413–420 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Petronilli, V. et al. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys. J. 76, 725–734 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Feldmann, G. et al. Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice. Hepatology 31, 674–683 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Wakabayashi, T. Structural changes of mitochondria related to apoptosis: swelling and megamitochondria formation. Acta Biochim. Pol. 46, 223–237 (1999).

    Article  CAS  PubMed  Google Scholar 

  30. Kaasik, A., Safiulina, D., Zharkovsky, A. & Veksler, V. Regulation of mitochondrial matrix volume. Am. J. Phys. 292, C157–C163 (2007).

    Article  CAS  Google Scholar 

  31. Song, W. et al. Assessing mitochondrial morphology and dynamics using fluorescence wide-field microscopy and 3D image processing. Methods 46, 295–303 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Leonard, A.P. et al. Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning. Biochim. Biophys. Acta 1853, 348–360 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. Reis, Y. et al. Multi-parametric analysis and modeling of relationships between mitochondrial morphology and apoptosis. PLoS One 7, e28694 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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).

    Article  CAS  PubMed  Google Scholar 

  35. Loew, L.M., Carrington, W., Tuft, R.A. & Fay, F.S. Physiological cytosolic Ca2+ transients evoke concurrent mitochondrial depolarizations. Proc. Natl. Acad. Sci. USA 91, 12579–12583 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Haworth, R.A. & Hunter, D.R. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch. Biochem. Biophys. 195, 460–467 (1979).

    Article  CAS  PubMed  Google Scholar 

  37. Takeyama, N., Matsuo, N. & Tanaka, T. Oxidative damage to mitochondria is mediated by the Ca(2+)-dependent inner-membrane permeability transition. Biochem. J. 294, 719–725 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kowaltowski, A.J., Castilho, R.F. & Vercesi, A.E. Mitochondrial permeability transition and oxidative stress. FEBS Lett. 495, 12–15 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Petronilli, V. et al. The voltage sensor of the mitochondrial permeability transition pore is tuned by the oxidation-reduction state of vicinal thiols. Increase of the gating potential by oxidants and its reversal by reducing agents. J. Biol. Chem. 269, 16638–16642 (1994).

    Article  CAS  PubMed  Google Scholar 

  40. Bravo, C., Chavez, E., Rodriguez, J.S. & Moreno-Sanchez, R. The mitochondrial membrane permeability transition induced by inorganic phosphate or inorganic arsenate. A comparative study. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 117, 93–99 (1997).

    Article  CAS  PubMed  Google Scholar 

  41. Kowaltowski, A.J., Castilho, R.F. & Vercesi, A.E. Opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of Ca2+ is dependent on mitochondrial-generated reactive oxygen species. FEBS Lett. 378, 150–152 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Wieckowski, M.R., Brdiczka, D. & Wojtczak, L. Long-chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore. FEBS Lett. 484, 61–64 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Beutner, G., Ruck, A., Riede, B., Welte, W. & Brdiczka, D. Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett. 396, 189–195 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Pfeiffer, D.R., Gudz, T.I., Novgorodov, S.A. & Erdahl, W.L. The peptide mastoparan is a potent facilitator of the mitochondrial permeability transition. J. Biol. Chem. 270, 4923–4932 (1995).

    Article  CAS  PubMed  Google Scholar 

  45. Li, J., Wang, J. & Zeng, Y. Peripheral benzodiazepine receptor ligand, PK11195 induces mitochondria cytochrome c release and dissipation of mitochondria potential via induction of mitochondria permeability transition. Eur. J. Pharmacol. 560, 117–122 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Vianello, A. et al. The mitochondrial permeability transition pore (PTP)-an example of multiple molecular exaptation? Biochim. Biophys. Acta 1817, 2072–2086 (2012).

    Article  CAS  PubMed  Google Scholar 

  47. Rizzuto, R., Brini, M., Pizzo, P., Murgia, M. & Pozzan, T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr. Biol. 5, 635–642 (1995).

    Article  CAS  PubMed  Google Scholar 

  48. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Di Virgilio, F., Fasolato, C. & Steinberg, T.H. Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells. Biochem. J. 256, 959–963 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Crompton, M., Ellinger, H. & Costi, A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem. J. 255, 357–360 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Baines, C.P. et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434, 658–662 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Kroemer, G., Galluzzi, L. & Brenner, C. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87, 99–163 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).

    Article  PubMed  CAS  Google Scholar 

  54. Sarkar, A.R. et al. Red emissive two-photon probe for real-time imaging of mitochondria trafficking. Anal. Chem. 86, 5638–5641 (2014).

    Article  CAS  PubMed  Google Scholar 

  55. Morozova, K.S. et al. MFar-red fluorescent protein excitable with red lasers for flow cytometry and superresolution STED nanoscopy. Biophys. J. 99, L13–L15 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Dumas, J.F. et al. Effect of transient and permanent permeability transition pore opening on NAD(P)H localization in intact cells. J. Biol. Chem. 284, 15117–15125 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Bejarano, I. et al. Role of calcium signals on hydrogen peroxide-induced apoptosis in human myeloid HL-60 Cells. Int. J. Biomed. Sci. 5, 246–256 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Deniaud, A. et al. Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene 27, 285–299 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Gerasimenko, J.V. et al. Menadione-induced apoptosis: roles of cytosolic Ca(2+) elevations and the mitochondrial permeability transition pore. J. Cell Sci. 115, 485–497 (2002).

    Article  CAS  PubMed  Google Scholar 

  60. Loor, G. et al. Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis. Free Radic. Biol. Med. 49, 1925–1936 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Novgorodov, S.A. & Gudz, T.I. Ceramide and mitochondria in ischemic brain injury. Int. J. Biochem. Mol. Biol. 2, 347–361 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Parra, V. et al. Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis. Cardiovasc. Res. 77, 387–397 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Salvioli, S., Ardizzoni, A., Franceschi, C. & Cossarizza, A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77–82 (1997).

    Article  CAS  PubMed  Google Scholar 

  65. Ohno, R., Koie, K., Kamiya, T., Kawashima, K. & Ishiguro, J. [Treatment of pulmonary infections probably caused by fungi in patients with acute leukemia with chlotrimazole (author's transl.)]. Rinsho Ketsueki 17, 876–883 (1976).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

P.P. is grateful to Camilla degli Scrovegni for continuous support. This research was supported by the Italian Ministry of Education, University and Research (COFIN no. 20129JLHSY_002, FIRB no. RBAP11FXBC_002, and Futuro in Ricerca no. RBFR10EGVP_001), local funds from the University of Ferrara and the Italian Ministry of Health to P.P. and C.G., Telethon (GGP15219/B), the Italian Association for Cancer Research (IG-14442 and MFAG-13521 to P.P. and C.G.), and the Italian Cystic Fibrosis Research Foundation (19/2014) to P.P. M.R.W. was supported by the National Science Centre, Poland (grant 2014/15/B/NZ1/00490), grant W100/HFSC/2011, and HFSP grant RGP0027/2011.

Author information

Authors and Affiliations

Authors

Contributions

M.B., C.M., G.M., C.G., M.R.W., and P.P. contributed extensively to the writing of this paper. M.B., G.M., and C.M. performed the experiments, analyzed data, and generated visual guides.

Corresponding author

Correspondence to Paolo Pinton.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Typical improper results

Example of typical experimental artifacts in the Co2+-calcein (A), mitochondrial membrane potential (B) and mitochondrial morphology assays (C). For solutions to these problem, refer to Table 2.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bonora, M., Morganti, C., Morciano, G. et al. Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques. Nat Protoc 11, 1067–1080 (2016). https://doi.org/10.1038/nprot.2016.064

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.064

This article is cited by

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