Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin

Atsushi Miyawaki, Juan Llopis, Roger Heim, J. Michael McCaffery, Joseph A. Adams, Mitsuhiko Ikura and Roger Y. Tsien
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Atsushi Miyawaki: University of California
Juan Llopis: University of California
Roger Heim: University of California
J. Michael McCaffery: University of California
Joseph A. Adams: San Diego State University
Mitsuhiko Ikura: Ontario Cancer Institute, University of Toronto
Roger Y. Tsien: University of California

Nature, 1997, vol. 388, issue 6645, 882-887

Abstract: 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)1,2, calmodulin3,4,5, the calmodulin-binding peptide M13 (ref. 6), and an enhanced green- or yellow-emitting GFP7,8,9. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs2. 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 µM at rest, and 1 to 50 µM 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.

Date: 1997
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DOI: 10.1038/42264

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