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Fluorescent Indicators for Calcium Based on Green Fluorescent Proteins (GFPs) and Calmodulin

Published online by Cambridge University Press:  02 July 2020

A. Miyawaki ,
J. Llopis ,
R. Heim ,
J.M. McCafFery ,
J.A. Adams ,
M. Ikura ,
H. Fujisaki ,
G.Y. Fan ,
M.H. Ellisman  and
R.Y. Tsien
A. Miyawaki
Affiliation:
Pharmacology Dept., San Diego, La Jolla, CA, 92093-0647
J. Llopis
Affiliation:
Pharmacology Dept., San Diego, La Jolla, CA, 92093-0647
R. Heim
Affiliation:
Pharmacology Dept., San Diego, La Jolla, CA, 92093-0647 Howard Hughes Medical Inst., San Diego, La Jolla, CA, 92093-0647
J.M. McCafFery
Affiliation:
Cellular and Molecular Medicine Div., La Jolla, CA, 92093-0647
J.A. Adams
Affiliation:
Chemistry Dept., San Diego State Univ., San Diego, CA, 92182-1030
M. Ikura
Affiliation:
Molecular and Structural Biol. Div., Ontario Cancer Institute, and Medical Biophysics Dept., University of Toronto, Toronto, M5G 2M9, Canada
H. Fujisaki
Affiliation:
Natl. Center for Microscopy and Imaging Res., Neurosciences Dept., Univ. of California, San Diego, La Jolla, CA, 92093-0647
G.Y. Fan
Affiliation:
Natl. Center for Microscopy and Imaging Res., Neurosciences Dept., Univ. of California, San Diego, La Jolla, CA, 92093-0647
M.H. Ellisman
Affiliation:
Natl. Center for Microscopy and Imaging Res., Neurosciences Dept., Univ. of California, San Diego, La Jolla, CA, 92093-0647
R.Y. Tsien
Affiliation:
Pharmacology Dept., San Diego, La Jolla, CA, 92093-0647 Howard Hughes Medical Inst., San Diego, La Jolla, CA, 92093-0647
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Extract

Cytosolic and organellar free Ca2+ concentrations are very dynamic; they 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.

Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jelly fish Aequorea victoria. Its cDNA can be concatenated with those encoding many other proteins, and the resulting fusion proteins are usually fluorescent and often preserve the biochemical functions and cellular localizations of the partner proteins. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation or emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET).

Our new indicators consist of tandem fusions of a blue- or cyan-emitting mutant of GFP, calmodulin (CaM), the calmodulin-binding peptide Ml3, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the Ml3 domain, increasing the FRET between the flanking GFPs (Fig. 1).

Type
Unique Approaches in Imaging, Computation and Communication for Characterization of the 3D Cell & Organelles I
Information
Microscopy and Microanalysis , Volume 4 , Issue S2: Proceedings: Microscopy & Microanalysis '98, Microscopy Society of America 56th Annual Meeting, Microbeam Analysis Society 32nd Annual Meeting, Atlanta, Georgia July 12-16, 1998 , July 1998 , pp. 446 - 447
Copyright
Copyright © Microscopy Society of America

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References

References:

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