Exploiting Cameleon Probes to Investigate Organelles Ca2+ Handling

  • Luisa Galla
  • Paola Pizzo
  • Elisa GreottiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)


Calcium ion (Ca2+) is a ubiquitous intracellular messenger able to generate versatile intracellular signals that modulate a large variety of functions in virtually every cell type. Chemical and genetic biosensors, targeted to different subcellular compartments, have been developed and continuously improved to monitor Ca2+ dynamics in living cells. Here we describe the usage of Förster resonance energy transfer (FRET)-based Cameleon probes to investigate Ca2+ influx across the plasma membrane (PM) or Ca2+ release from the main intracellular Ca2+ store, the endoplasmic reticulum (ER).

Key words

Ca2+ signaling Ca2+ imaging Cameleon FRET SOCE Endoplasmic reticulum IP3 receptor 



This work was supported by Telethon GGP16029, the University of Padua, and the EU Joint Programme for Neurodegenerative Disease Research (JPND) CeBioND, supported by the MIUR (DM 9; 08/01/2015).


  1. 1.
    Lu KP, Means AR (1993) Regulation of the cell cycle by calcium and calmodulin. Endocr Rev 14:40–58CrossRefGoogle Scholar
  2. 2.
    Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat. Rev Mol Cell Biol Oct 1(1):11–21CrossRefGoogle Scholar
  3. 3.
    Orrenius S, Zhivotovsky B, Nicotera P (2003) Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4:552–565CrossRefGoogle Scholar
  4. 4.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529CrossRefGoogle Scholar
  5. 5.
    Clapham DE (2007) Calcium signaling. Cell 131(6):1047–1058CrossRefGoogle Scholar
  6. 6.
    Chemaly ER, Troncone L, Lebeche D (2018) SERCA control of cell death and survival. Cell Calcium 69:46–61CrossRefGoogle Scholar
  7. 7.
    Alonso MT, Rodríguez-Prados M, Navas-Navarro P, Rojo-Ruiz J, García-Sancho J (2017) Using aequorin probes to measure Ca2+ in intracellular organelles. Cell Calcium 64:3–11CrossRefGoogle Scholar
  8. 8.
    Pizzo P, Lissandron V, Capitanio P, Pozzan T (2011) Ca(2+) signalling in the Golgi apparatus. Cell Calcium 50(2):184–192CrossRefGoogle Scholar
  9. 9.
    Brini M, Murgia M, Pasti L, Picard D, Pozzan T, Rizzuto R (1993) Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin. EMBO J 12:4813–4819CrossRefGoogle Scholar
  10. 10.
    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 388:882–887. CrossRefPubMedGoogle Scholar
  11. 11.
    Manjarrés IM, Chamero P, Domingo B, Molina F, Llopis J, Alonso MT, García-Sancho J (2008) Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles. Pflugers Arch 455:961–970. CrossRefPubMedGoogle Scholar
  12. 12.
    Bootman MD, Thomas D, Tovey SC, Berridge MJ, Lipp P (2000) Nuclear calcium signalling. Cell Mol Life Sci 57:371–378CrossRefGoogle Scholar
  13. 13.
    Rizzuto R, Simpson AW, Brini M, Pozzan T (1992) Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358:325–327. CrossRefPubMedGoogle Scholar
  14. 14.
    Rizzuto R, Brini M, Murgia M, Pozzan T (1993) Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 262:744–747CrossRefGoogle Scholar
  15. 15.
    Montero M, Alonso MT, Carnicero E, Cuchillo-Ibáñez I, Albillos A, García AG, García-Sancho J, Alvarez J (2000) Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion. Nat Cell Biol 2:57–61CrossRefGoogle Scholar
  16. 16.
    Putney JW (2017) Forms and functions of store-operated calcium entry mediators, STIM and Orai. Adv Biol Regul pii S2212-4926(17):30171–30179Google Scholar
  17. 17.
    Morgan AJ (2016) Ca2+ dialogue between acidic vesicles and ER. Biochem Soc Trans 44(2):546–553CrossRefGoogle Scholar
  18. 18.
    Pendin D, Greotti E, Lefkimmiatis K, Pozzan T (2017) Exploring cells with targeted biosensors. J Gen Physiol 149(1):1–36CrossRefGoogle Scholar
  19. 19.
    Baird GS, Zacharias DA, Tsien RY (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci U S A 96:11241–11246CrossRefGoogle Scholar
  20. 20.
    Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. J Biol Chem 276:29188–29194CrossRefGoogle Scholar
  21. 21.
    Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat Biotechnol 19:137–141CrossRefGoogle Scholar
  22. 22.
    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 98:3197–3202CrossRefGoogle Scholar
  23. 23.
    Horikawa K, Yamada Y, Matsuda T, Kobayashi K, Hashimoto M, Matsu-ura T, Miyawaki A, Michikawa T, Mikoshiba K, Nagai T (2010) Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano. Nat Methods 7(9):729–732CrossRefGoogle Scholar
  24. 24.
    Heim N, Griesbeck O (2004) Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein. J Biol Chem 279:14280–14286CrossRefGoogle Scholar
  25. 25.
    Palmer AE, Tsien RY (2006) Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1:1057–1065CrossRefGoogle Scholar
  26. 26.
    Greotti E, Wong A, Pozzan T, Pendin D, Pizzo P (2016) Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER. Sensors 16:1419. CrossRefGoogle Scholar
  27. 27.
    Wong AK, Capitanio P, Lissandron V, Bortolozzi M, Pozzan T, Pizzo P (2013) Heterogeneity of Ca2+ handling among and within Golgi compartments. J Mol Cell Biol 5(4):266–276CrossRefGoogle Scholar
  28. 28.
    Lissandron V, Podini P, Pizzo P, Pozzan T (2010) Unique characteristics of Ca2+ homeostasis of the trans-Golgi compartment. Proc Natl Acad Sci U S A 107(20):9198–9203CrossRefGoogle Scholar
  29. 29.
    Drago I, Giacomello M, Pizzo P, Pozzan T (2008) Calcium dynamics in the peroxisomal lumen of living cells. J Biol Chem 283(21):14384–14390CrossRefGoogle Scholar
  30. 30.
    Gordon GW, Berry G, Liang XH, Levine B, Herman B (1998) Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J 74:2702–2713.30CrossRefGoogle Scholar
  31. 31.
    Penner R, Fasolato C, Hoth M (1993) Calcium influx and its control by calcium release. Curr Opin Neurobiol 3(3):368–374CrossRefGoogle Scholar
  32. 32.
    Zal T, Gascoigne NR (2004) Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 86:3923–3939CrossRefGoogle Scholar

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

Authors and Affiliations

  1. 1.Department of Biomedical SciencesUniversity of PaduaPaduaItaly
  2. 2.Neuroscience Institute —Italian National Research Council (CNR)PaduaItaly

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