Abstract
Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Shimomura O, Johnson FH (1973) Chemical nature of the light emitter in bioluminescence of aequorin. Tetrahedron Lett 14:2963–2966. https://doi.org/10.1016/S0040-4039(01)96293-8
Charbonneau H, Walsh KA, McCann RO, Prendergast FG, Cormier MJ, Vanaman TC (1985) Amino acid sequence of the calcium-dependent photoprotein aequorin. Biochemistry 24:6762–6771
Head JF, Inouye S, Teranishi K, Shimomura O (2000) The crystal structure of the photoprotein aequorin at 2.3 A resolution. Nature 405:372–376
Shimomura O, Johnson FH (1975) Regeneration of the photoprotein aequorin. Nature 256:236–238
Granatiero V, Patron M, Tosatto A, Merli G, Rizzuto R (2014) The use of aequorin and its variants for Ca2+ measurements. Cold Spring Harb Protoc 2014:9–16
Brini M (2008) Calcium-sensitive photoproteins. Methods 46:160–166
Bianchi K, Rimessi A, Prandini A, Szabadkai G, Rizzuto R (2004) Calcium and mitochondria: mechanisms and functions of a troubled relationship. Biochim Biophys Acta Mol Cell Res 1742:119–131
Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T, Rizzuto R (1995) Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 14:5467–5475
Kendall JM, Sala-Newby G, Ghalaut V, Dormer RL, Cambell AK (1992) Engineering the Ca2+−activated photoprotein aequorin with reduced affinity for calcium. Biochem Biophys Res Commun 187:1091–1097
De la Fuente S, Fonteriz RI, de la Cruz PJ, Montero M, Alvarez J (2012) Mitochondrial free [Ca(2+)] dynamics measured with a novel low-Ca(2+) affinity aequorin probe. Biochem J 445:371–376
Bonora M, Giorgi C, Bononi A, Marchi S, Patergnani S, Rimessi A, Rizzuto R, Pinton P (2013) Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes. Nat Protoc 8(11):2105
Shimomura O, Musicki B, Kishi Y, Inouye S (1993) Light-emitting properties of recombinant semisynthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium 14:373–378
Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R (1995) Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+](c)). A critical evaluation. J Biol Chem 270(17):9896–9903
Brini M, Murgia M, Pasti L, Picard D, Pozzan T, Rizzuto R (1993) Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin. EMBO J 12:4813–4819
Brini M, Marsault R, Bastianutto C, Pozzan T, Rizzuto R (1994) Nuclear targeting of aequorin. A new approach for measuring nuclear Ca2+ concentration in intact cells. Cell Calcium 16:259–268
Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T, Rizzuto R (1995) Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 14:5467
Rizzuto R, Simpson AWM, Brini M, Pozzan T (1992) Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358:325–327
Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Tuft RA, Pozzan T (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280:1763–1766
Fliegel L, Newton E, Burns K, Michalak M (1990) Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum. J Biol Chem 265:15496–15502
Sitia R, Meldolesi J (1992) Endoplasmic reticulum: a dynamic patchwork of specialized subregions. Mol Biol Cell 3:1067–1072
Brini M, De Giorgi F, Murgia M, Marsault R, Massimino ML, Cantini M, Rizzuto R, Pozzant T (1997) Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes. Mol Biol Cell 8:129–143
Pinton P, Pozzan T, Rizzuto R (1998) The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum. EMBO J 17:5298
Mitchell KJ, Pinton P, Varadi A, Tacchetti C, Ainscow EK, Pozzan T, Rizzuto R, Rutter GA (2001) Dense core secretory vesicles revealed as a dynamic Ca2+ store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera. J Cell Biol 155:41
Lasorsa FM, Pinton P, Palmieri L, Scarcia P, Rottensteiner H, Rizzuto R, Palmieri F (2008) Peroxisomes as novel players in cell calcium homeostasis. J Biol Chem 283:15300–15308
Robert V, Pinton P, Tosello V, Rizzuto R, Pozzan T (2000) Recombinant aequorin as tool for monitoring calcium concentration in subcellular compartments. Methods Enzymol 327:440–456
Chiesa A, Rapizzi E, Tosello V, Pinton P, de Virgilio M, Fogarty KE, Rizzuto R (2001) Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling. Biochem J 355:1–12
Brini M, Marsault R, Bastianutto C, Alvarez J, Pozzan T, Rizzuto R (1995) Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). J Biol Chem 270:9896–9903
Ottolini D, Calì T, Brini M (2014) Methods to measure intracellular Ca2+ fluxes with organelle-targeted aequorin-based probes. Methods Enzymol 543:21–45
Ottolini D, Calì T, Brini M (2013) Measurements of Ca2+ concentration with recombinant targeted luminescent probes. In: Methods in molecular biology. Humana Press, Clifton, NJ, pp 273–291
Allen DG, Blinks JR (1978) Calcium transients in aequorin-injected frog cardiac muscle. Nature 273:509–513
Acknowledgments
We thank Anna Raffaello for the critical reading of the manuscript. This work was supported by the Italian Telethon Foundation (GGP16026) and the French Muscular Dystrophy Association (AFM-Téléthon) (19471).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Feno, S., Di Marco, G., De Mario, A., Monticelli, H., Reane, D.V. (2019). High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels. In: Raffaello, A., Vecellio Reane, D. (eds) Calcium Signalling. Methods in Molecular Biology, vol 1925. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9018-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9018-4_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9017-7
Online ISBN: 978-1-4939-9018-4
eBook Packages: Springer Protocols