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Assessment of Mitochondrial Ca2+ Uptake

  • András T. Deak
  • Claire Jean-Quartier
  • Alexander I. Bondarenko
  • Lukas N. Groschner
  • Roland Malli
  • Wolfgang F. Graier
  • Markus Waldeck-Weiermair
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1264)

Abstract

Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.

Key words

Mitochondrial Ca2+ uptake Calcium Green Fura-2 Rhod-2 FRET Oxidative phosphorylation Mitochondrial membrane potential Mitoplast Patch-clamp recording Ca2+ imaging 

References

  1. 1.
    Jouaville LS, Pinton P, Bastianutto C, Rutter GA, Rizzuto R (1999) Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming. Proc Natl Acad Sci U S A 96:13807–13812PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS (2004) Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 287(4):C817–C833PubMedCrossRefGoogle Scholar
  3. 3.
    Giorgi C, Romagnoli A, Pinton P, Rizzuto R (2008) Ca2+ signaling, mitochondria and cell death. Curr Mol Med 8:119–130PubMedCrossRefGoogle Scholar
  4. 4.
    De Stefani D, Raffaello A, Teardo E, Szabò I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476:336–340PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Mallilankaraman K, Cárdenas C, Doonan PJ, Chandramoorthy HC, Irrinki KM, Golenár T et al (2012) MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism. Nat Cell Biol 14:1336–1343PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y et al (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476:341–345PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, Palmer AE et al (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake. Nature 467:291–296PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Jiang D, Zhao L, Clapham DE (2009) Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter. Science 326:144–147PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Zecchini E, Siviero R, Giorgi C, Rizzuto R, Pinton P (2007) Mitochondrial calcium signalling: message of life and death. Ital J Biochem 56:235–242PubMedGoogle Scholar
  10. 10.
    Jean-Quartier C, Bondarenko AI, Alam MR, Trenker M, Waldeck-Weiermair M, Malli R et al (2010) Studying mitochondrial Ca2+ uptake—a revisit. Mol Cell Endocrinol 353:114–127CrossRefGoogle Scholar
  11. 11.
    Sancak Y, Markhard AL, Kitami T, Kovács-Bogdán E, Kamer KJ, Udeshi ND et al (2013) EMRE is an essential component of the mitochondrial calcium uniporter complex. Science 342:1379–1382PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Waldeck-Weiermair M, Deak AT, Groschner LN, Alam MR, Jean-Quartier C, Malli R et al (2013) Molecularly distinct routes of mitochondrial Ca2+ uptake are activated depending on the activity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). J Biol Chem 288:15367–15379PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Bright GR, Fisher GW, Rogowska J, Taylor DL (1989) Fluorescence ratio imaging microscopy. Methods Cell Biol 30:157–192PubMedCrossRefGoogle Scholar
  14. 14.
    Eberhard M, Erne P (1991) Calcium binding to fluorescent calcium indicators: calcium green, calcium orange and calcium crimson. Biochem Biophys Res Commun 180:209–215PubMedCrossRefGoogle Scholar
  15. 15.
    Rizzuto R, Simpson AW, Brini M, Pozzan T (1992) Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358:325–327PubMedCrossRefGoogle Scholar
  16. 16.
    Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A 98(6):3197–3202PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Nakai J, Ohkura M, Imoto KA (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol 19:137–141PubMedCrossRefGoogle Scholar
  18. 18.
    Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M et al (2011) An expanded palette of genetically encoded Ca2+ indicators. Science 333:1888–1891PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M et al (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887PubMedCrossRefGoogle Scholar
  20. 20.
    Palmer AE, Jin C, Reed JC, Tsien RY (2004) Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc Natl Acad Sci U S A 101:17404–17409PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Palmer AE, Giacomello M, Kortemme T, Hires SA, Lev-Ram V, Baker D et al (2006) Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol 13:521–530PubMedCrossRefGoogle Scholar
  22. 22.
    McCombs JE, Palmer AE (2008) Measuring calcium dynamics in living cells with genetically encodable calcium indicators. Methods 46:152–159PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Carlson HJ, Campbell RE (2009) Genetically encoded FRET-based biosensors for multiparameter fluorescence imaging. Curr Opin Biotechnol 20:19–27PubMedCrossRefGoogle Scholar
  24. 24.
    Waldeck-Weiermair M, Alam MR, Khan MJ, Deak AT, Vishnu N, Karsten F et al (2012) Spatiotemporal correlations between cytosolic and mitochondrial Ca2+ signals using a novel red-shifted mitochondrial targeted cameleon. PLoS One 7:e45917PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Bondarenko AI, Jean-Quartier C, Malli R, Graier WF (2013) Characterization of distinct single-channel properties of Ca2+ inward currents in mitochondria. Pflugers Arch 465:997–1010PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Wiederkehr A, Szanda G, Akhmedov D, Mataki C, Heizmann CW, Schoonjans K et al (2011) Mitochondrial matrix calcium is an activating signal for hormone secretion. Cell Metab 13:601–611PubMedCrossRefGoogle Scholar
  27. 27.
    Vishnu N, Jadoon Khan M, Karsten F, Groschner LN, Waldeck-Weiermair M, Rost R et al (2014) ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+-release. Mol Biol Cell 25(3):368–379PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Nakano M, Imamura H, Nagai T, Noji H (2011) Ca2+ regulation of mitochondrial ATP synthesis visualized at the single cell level. ACS Chem Biol 6:709–715PubMedCrossRefGoogle Scholar
  29. 29.
    Fieni F, Lee SB, Jan YN, Kirichok Y (2012) Activity of the mitochondrial calcium uniporter varies greatly between tissues. Nat Commun 3:1317PubMedCrossRefGoogle Scholar
  30. 30.
    Fedorenko A, Lishko PV, Kirichok Y (2012) Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria. Cell 151:400–413PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Decker GL, Greenawalt JW (1977) Ultrastructural and biochemical studies of mitoplasts and outer membranes derived from French-pressed mitochondria. Advances in mitochondrial subfractionation. J Ultrastruct Res 59:44–56PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • András T. Deak
    • 1
  • Claire Jean-Quartier
    • 1
  • Alexander I. Bondarenko
    • 1
  • Lukas N. Groschner
    • 1
  • Roland Malli
    • 1
  • Wolfgang F. Graier
    • 1
  • Markus Waldeck-Weiermair
    • 1
  1. 1.Institute of Molecular Biology and Biochemistry, Center of Molecular MedicineMedical University of GrazGrazAustria

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