Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.
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.
Miyawaki
A
A
Department of Pharmacology, University of California, San Diego, La Jolla 92093-0647, USA.
Llopis
J
J
Heim
R
R
McCaffery
J M
JM
Adams
J A
JA
Ikura
M
M
Tsien
R Y
RY
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
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