부산대학교 도서관

Important Ca sup 2+ 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 Ca+ 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 Ca+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs . Calmodulin mutations can tune the Ca sup 2+ affinities to measure free Ca+ concentrations in the range 10 sup -8 to 10 sup -2 M. We have visualized free Ca+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca+ concentration in the endosplasmic reticulum of individual cells ranged from 60 to 400 micro M at rest, and 1 to 50 micro M after Ca+ 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 Ca+ signals and protein heterodimerization in individual live cells.