Measuring Ca2+ Levels in Subcellular Compartments with Genetically Encoded GFP-Based Indicators

  • Mattia Vicario
  • Tito CalìEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)


Ca2+ homeostasis is crucial for the entire life of eukaryotic cells from the beginning to the end. Mishandling in Ca2+ homeostasis is indeed linked with a large number of pathological conditions. Thus, the possibility to specifically monitor cellular calcium fluxes in different subcellular compartments represents a key tool to deeply understand the mechanisms involved in cellular dysfunctions. To cope with this need, several Ca2+ indicators have been developed allowing to accurately measure both basal Ca2+ concentration and agonist-induced Ca2+ signals in a wide spectrum of organelles. Among these, the genetically encoded GFP-based indicators are routinely used to measure Ca2+ transients thanks to their ability to change their spectral properties in response to Ca2+ binding. In this chapter, we will describe a protocol that utilizes the GCaMP6f probe targeted to mitochondria (4mtGCaMP) to measure mitochondrial calcium levels in resting conditions in HeLa cells. This method allows to easily and quickly register alterations of mitochondrial Ca2+ homeostasis in different cell populations and experimental settings, representing a precious tool to unravel the pathological pathways leading to pathogenic conditions.

Key words

Calcium Fluorescent indicator Calcium handling GFP GCaMP 


  1. 1.
    Carafoli E, Malmstrom K, Sigel E, Crompton M (1976) The regulation of intracellular calcium. Clin Endocrinol 5 Suppl:49S–59SCrossRefGoogle Scholar
  2. 2.
    Brini M, Cali T, Ottolini D, Carafoli E (2013) Intracellular calcium homeostasis and signaling. Metal Ions Life Sci 12:119–168CrossRefGoogle Scholar
  3. 3.
    Prins D, Michalak M (2011) Organellar calcium buffers. Cold Spring Harb Perspect Biol 3(3)CrossRefGoogle Scholar
  4. 4.
    Schwaller B (2010) Cytosolic Ca2+ buffers. Cold Spring Harb Perspect Biol 2(11):a004051CrossRefGoogle Scholar
  5. 5.
    Foskett JK (2010) Inositol trisphosphate receptor Ca2+ release channels in neurological diseases. Pflugers Arch 460(2):481–494CrossRefGoogle Scholar
  6. 6.
    Camandola S, Mattson MP (2011) Aberrant subcellular neuronal calcium regulation in aging and Alzheimer’s disease. Biochim Biophys Acta 1813(5):965–973CrossRefGoogle Scholar
  7. 7.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887CrossRefGoogle Scholar
  8. 8.
    Romoser VA, Hinkle PM, Persechini A (1997) Detection in living cells of Ca2+−dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 272(20):13270–13274CrossRefGoogle Scholar
  9. 9.
    Miyawaki A, Nagai T, Mizuno H (2013) Imaging intracellular free Ca2+ concentration using yellow cameleons. Cold Spring Harb Protoc 2013(11)CrossRefGoogle Scholar
  10. 10.
    Palmer AE, Tsien RY (2006) Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1(3):1057–1065CrossRefGoogle Scholar
  11. 11.
    Tian L, Hires SA, Looger LL (2012) Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harb Protoc 2012(6):647–656CrossRefGoogle Scholar
  12. 12.
    Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, Campbell RE (2011) An expanded palette of genetically encoded Ca(2)(+) indicators. Science 333(6051):1888–1891CrossRefGoogle Scholar
  13. 13.
    Akerboom J, Carreras Calderon N, Tian L, Wabnig S, Prigge M, Tolo J, Gordus A, Orger MB, Severi KE, Macklin JJ, Patel R, Pulver SR, Wardill TJ, Fischer E, Schuler C, Chen TW, Sarkisyan KS, Marvin JS, Bargmann CI, Kim DS, Kugler S, Lagnado L, Hegemann P, Gottschalk A, Schreiter ER, Looger LL (2013) Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neurosci 6:2CrossRefGoogle Scholar
  14. 14.
    Carlson HJ, Campbell RE (2013) Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry. Protein Eng Des Sel 26(12):763–772CrossRefGoogle Scholar
  15. 15.
    Csordas G, Varnai P, Golenar T, Roy S, Purkins G, Schneider TG, Balla T, Hajnoczky G (2010) Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface. Mol Cell 39(1):121–132CrossRefGoogle Scholar
  16. 16.
    Liu S, He J, Jin H, Yang F, Lu J, Yang J (2011) Enhanced dynamic range in a genetically encoded Ca2+ sensor. Biochem Biophys Res Commun 412(1):155–159CrossRefGoogle Scholar
  17. 17.
    Waldeck-Weiermair M, Alam MR, Khan MJ, Deak AT, Vishnu N, Karsten F, Imamura H, Graier WF, Malli R (2012) Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon. PLoS One 7(9):e45917CrossRefGoogle Scholar
  18. 18.
    Heim N, Griesbeck O (2004) Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein. J Biol Chem 279(14):14280–14286CrossRefGoogle Scholar
  19. 19.
    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):3197–3202CrossRefGoogle Scholar
  20. 20.
    Lu X, Ginsburg KS, Kettlewell S, Bossuyt J, Smith GL, Bers DM (2013) Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release. Circ Res 112(3):424–431CrossRefGoogle Scholar
  21. 21.
    Hasan MT, Friedrich RW, Euler T, Larkum ME, Giese G, Both M, Duebel J, Waters J, Bujard H, Griesbeck O, Tsien RY, Nagai T, Miyawaki A, Denk W (2004) Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol 2(6):e163CrossRefGoogle Scholar
  22. 22.
    Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276(31):29188–29194CrossRefGoogle Scholar
  23. 23.
    Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499(7458):295–300CrossRefGoogle Scholar
  24. 24.
    Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J, Xin HB, Sanbe A, Gulick J, Mathai J, Robbins J, Salama G, Nakai J, Kotlikoff MI (2006) Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci U S A 103(12):4753–4758CrossRefGoogle Scholar
  25. 25.
    Bianchi K, Rimessi A, Prandini A, Szabadkai G, Rizzuto R (2004) Calcium and mitochondria: mechanisms and functions of a troubled relationship. Biochim Biophys Acta 1742(1-3):119–131CrossRefGoogle Scholar
  26. 26.
    Thomas D, Tovey SC, Collins TJ, Bootman MD, Berridge MJ, Lipp P (2000) A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28(4):213–223CrossRefGoogle Scholar
  27. 27.
    Jimenez-Moreno R, Wang ZM, Messi ML, Delbono O (2010) Sarcoplasmic reticulum Ca2+ depletion in adult skeletal muscle fibres measured with the biosensor D1ER. Pflugers Arch 459(5):725–735CrossRefGoogle Scholar
  28. 28.
    Mizuno H, Sassa T, Higashijima S, Okamoto H, Miyawaki A (2013) Transgenic zebrafish for ratiometric imaging of cytosolic and mitochondrial Ca2+ response in teleost embryo. Cell Calcium 54(3):236–245CrossRefGoogle Scholar
  29. 29.
    Yu D, Baird GS, Tsien RY, Davis RL (2003) Detection of calcium transients in drosophila mushroom body neurons with camgaroo reporters. J Neurosci 23(1):64–72CrossRefGoogle Scholar
  30. 30.
    Akerboom J, Chen TW, Wardill TJ, Tian L, Marvin JS, Mutlu S, Calderon NC, Esposti F, Borghuis BG, Sun XR, Gordus A, Orger MB, Portugues R, Engert F, Macklin JJ, Filosa A, Aggarwal A, Kerr RA, Takagi R, Kracun S, Shigetomi E, Khakh BS, Baier H, Lagnado L, Wang SS, Bargmann CI, Kimmel BE, Jayaraman V, Svoboda K, Kim DS, Schreiter ER, Looger LL (2012) Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci 32(40):13819–13840CrossRefGoogle Scholar
  31. 31.
    De Stefani D, Raffaello A, Teardo E, Szabo I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476(7360):336–340CrossRefGoogle Scholar
  32. 32.
    Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, Palmer AE, Mootha VK (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake. Nature 467(7313):291–296CrossRefGoogle Scholar
  33. 33.
    Patron M, Checchetto V, Raffaello A, Teardo E, Vecellio Reane D, Mantoan M, Granatiero V, Szabo I, De Stefani D, Rizzuto R (2014) MICU1 and MICU2 finely tune the mitochondrial Ca Uniporter by exerting opposite effects on MCU activity. Mol Cell 53(5):726–737CrossRefGoogle Scholar
  34. 34.
    Logan CV, Szabadkai G, Sharpe JA, Parry DA, Torelli S, Childs AM, Kriek M, Phadke R, Johnson CA, Roberts NY, Bonthron DT, Pysden KA, Whyte T, Munteanu I, Foley AR, Wheway G, Szymanska K, Natarajan S, Abdelhamed ZA, Morgan JE, Roper H, Santen GW, Niks EH, van der Pol WL, Lindhout D, Raffaello A, De Stefani D, den Dunnen JT, Sun Y, Ginjaar I, Sewry CA, Hurles M, Rizzuto R, Consortium UK, Duchen MR, Muntoni F, Sheridan E (2014) Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling. Nat Genet 46(2):188–193CrossRefGoogle Scholar
  35. 35.
    Vecellio Reane D, Vallese F, Checchetto V, Acquasaliente L, Butera G, De Filippis V, Szabo I, Zanotti G, Rizzuto R, Raffaello A (2016) A MICU1 splice variant confers high sensitivity to the mitochondrial Ca(2+) uptake machinery of skeletal muscle. Mol Cell 64(4):760–773CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biomedical SciencesUniversity of PaduaPaduaItaly

Personalised recommendations