title: Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.
abstract: Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-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.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla 92093-0647, USA.
authors.forename: A
authors.initials: A
authors.lastname: Miyawaki
authors.forename: J
authors.initials: J
authors.lastname: Llopis
authors.forename: R
authors.initials: R
authors.lastname: Heim
authors.forename: J M
authors.initials: JM
authors.lastname: McCaffery
authors.forename: J A
authors.initials: JA
authors.lastname: Adams
authors.forename: M
authors.initials: M
authors.lastname: Ikura
authors.forename: R Y
authors.initials: RY
authors.lastname: Tsien
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000032:analysis* / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry; D004735:Energy Transfer; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D002147:Calmodulin; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium