Nature © Macmillan Publishers Ltd 1997Fluorescent indicators for Ca2+based on green fluorescentproteins and calmodulinAtsushi Miyawaki*, Juan Llopis*, Roger Heim*†,J. Michael McCaffery‡, Joseph A. Adams§,Mitsuhiko Ikurak, & Roger Y. Tsien*†* Department of Pharmacology,†Howard Hughes Medical Institute, and‡Division of Cellular and Molecular Medicine, University of California,San Diego, La Jolla, California 92093-0647, USA§ Department of Chemistry, San Diego State University, San Diego,California 92182-1030, USAk Division of Molecular and Structural Biology, Ontario Cancer Institute, andDepartment of Medical Biophysics, University of Toronto, Toronto M5G 2M9,Canada, and Center for Tsukuba Advanced Research Alliance,University of Tsukuba, Tsukuba 305, Japan.........................................................................................................................Important Ca2+signals in the cytosol and organelles are oftenextremely localized and hard to measure. To overcome thisproblem we have constructed new fluorescent indicators forCa2+that are genetically encoded without cofactors and aretargetable to specific intracellular locations. We have dubbedthese fluorescent indicators ‘cameleons’. They consist of tandemfusions of a blue- or cyan-emitting mutant of the green fluores-cent protein (GFP)1,2, calmodulin3–5, the calmodulin-bindingpeptide M13 (ref. 6), and an enhanced green- or yellow-emittingGFP7–9. Binding of Ca2+makes calmodulin wrap around the M13domain, increasing the fluorescence resonance energy transfer(FRET) between the flanking GFPs2. Calmodulin mutations cantune the Ca2+affinities to measure free Ca2+concentrations in therange 10−8to 10−2M. We have visualized free Ca2+dynamics in thecytosol, nucleus and endoplasmic reticulum of single HeLa cellstransfected with complementary DNAs encoding chimaeras bear-ing appropriate localization signals. Ca2+concentration in theendoplasmic reticulum of individual cells ranged from 60 to400 mM at rest, and 1 to 50 mM after Ca2+mobilization. FRET isalso an indicator of the reversible intermolecular association ofcyan-GFP-labelled calmodulin with yellow-GFP-labelled M13.Thus FRET between GFP mutants can monitor localized Ca2+signals and protein heterodimerization in individual live cells.Cytosolic and organellar free Ca2+concentrations are among themost important and dynamic intracellular signals, and are usuallymeasured using synthetic fluorescent chelators10–13or recombinantaequorin14,15. The chelators are easily imaged but are difficult totarget precisely to specific intracellular locations, whereas aequorinis easily targeted but requires the incorporation of coelenterazine, isirreversibly consumed by Ca2+, and is very difficult to image becauseits luminescence produces less than one photon per molecule. Tocombine the brightness of fluorescent indicators with the target-ability of a biosynthetic indicator, we have used GFP, a sponta-neously fluorescent protein from the jellyfish Aequorea victoria. ItscDNA can be concatenated with those encoding many otherproteins, and the resulting fusion proteins are usually fluorescentand often preserve the biochemical functions and cellular localiza-tions of the partner proteins. Mutagenesis has produced GFPmutants with shifted wavelengths of excitation or emission1,2,7,9that can serve as donors and acceptors for FRET. FRET is a non-destructive spectroscopic method that can monitor the proximityand relative angular orientation of fluorophores in living cells. Thedonor and acceptor fluorophores can be entirely separate orletters to nature882 NATURE|VOL 388|28 AUGUST 1997GFP or YFPCaMM13440 or 480 nmCaM510 or 535 nmFRET4 Ca2+-Ca2++4 Ca2+BFP or CFPBFP or CFPGFP or YFP370 or 440 nm370 or 440 nmaFigure 1 Schematic structures and sequences of cameleons. a, Schemeshowing how FRET between GFPs can measure Ca2+. The GFPs are drawn assimple rigid cylinders, reflecting their crystal structures9. Schematic structures ofcalmodulin (CaM) without Ca2+, disordered unbound M13, and the Ca2+–calmo-dulin–M13 complex are derived from crystallography4, NMR6and modelling16, butthe relative orientations of the GFPs are unknown. b, Domain structure ofcameleons expressed in bacteria for in vitro characterization, showingsequences of the boundaries between the donor GFP and Xenopus calmodulin(XCaM) and between M13 and the acceptor GFP. c, Consitition of cameleons forexpression and imaging in mammalian cells. CRsig, calreticulin signal sequence,MLLSVPLLLGLLGLAAAD24; nls, nuclear localizaiton signal, PKKKRKVEDA;KDEL, ER retention signal24; kz, Kozak consensus sequence for optimal transla-tional iniitation in mammalian cells28. The significant mutations in the four GFPsare: EBFP, F64L/Y66H/Y1452; EGFP, F64L/S65T8; ECFP, F64L/S65T/Y66W/N146I/M153T/V163A/N212K2; and EYFP, S65G/S72A/T203Y9.Blue or Cyan mutant GFPXCaMGreen or Yellow mutant GFPGlyGlyCaM-M13Ala Ala Arg Met His AspGCT GCT CGC ATG CAT GAC226 2273Sph IIle Ser Glu Leu Met SerATC TCC GAG CTC ATG AGT20 21 1 2Sac IAla Leu Glu Leu Met SerGCA CTG GAG CTC ATG AGT25 26 1 2Sac IPolyhistidinetagcameleon yellowcameleonbEBFPEGFPkzcameleon-2EBFPEGFPkznlscameleon-2nuECFPEYFPCRsigkzKDELyellow cameleon-4erE31QECFPEYFPkz yellow cameleon-2ECFPEYFPkzkzM13CaMECFP-CaM and M13-EYFPCaM-M13ECFPEYFPKDELCRsigkzE104Q yellow cameleon-3erEBFPEGFPKDELCRsigkzE104Qcameleon-3ercNature © Macmillan Publishers Ltd 1997attached to the same macromolecule. In the latter case, ligand-induced conformational changes might be monitored by FRET ifthe amino and carboxy termini of the binding protein are fused to adonor and acceptor GFP. This approach has several advantages overthe usual covalent labelling with fluorescent probes. First, theindicator is generated in situ by gene transfer into the cells ororganisms, obviating large-scale expression, purification, labellingand microinjection of recombinant proteins that must be soluble.Second, the sites of fusions are defined exactly, giving a molecularlyhomogenous product without the use of elaborate protein chem-istry. Third, the chromophore of GFP is fixed in the protein9. If theGFP donor and acceptor are rigidly fused to a host protein, minorchanges in the relative orientation of the ends of the latter wouldalter FRET. In contrast, most conventional fluorescent labels areattached by flexible linkers that at least partly decouple the fluor-ophore orientation from that of the protein to which it is