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)(1,2), calmodulin(3-5), the calmodulin-binding peptide M13 (ref, 6), and an enhanced green-or yellow-emitting GFP(7-9). Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs(2), 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 mu M at rest, and 1 to 50 mu M 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.