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Imaging Protein Interactions and Gene Expression in Individual Cells by Fluorescence Resonance Energy Transfer

Published online by Cambridge University Press:  02 July 2020

R.Y. Tsien ,
A. Miyawaki ,
R. Kerr ,
G. Baird ,
B.A. Griffin ,
S.R. Adams ,
G. Zlokarnik ,
M. Whitney ,
P.A. Negulescu  and
W.-H. Li
R.Y. Tsien
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
A. Miyawaki
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
R. Kerr
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
G. Baird
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
B.A. Griffin
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
S.R. Adams
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
G. Zlokarnik
Affiliation:
Aurora Biosciences Corp., 11010 Torreyana Rd., San Diego, CA92121, USA.
M. Whitney
Affiliation:
Aurora Biosciences Corp., 11010 Torreyana Rd., San Diego, CA92121, USA.
P.A. Negulescu
Affiliation:
Aurora Biosciences Corp., 11010 Torreyana Rd., San Diego, CA92121, USA.
W.-H. Li
Affiliation:
Howard Hughes Medical Institute and Depts. of Pharmacology and Chemistry & Biochemistry, University of California San Diego, La Jolla, CA92093-0647
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Extract

Interactions between proteins or protein domains can be imaged by fusing them to cyan (CFP) and yellow (YFP) mutants of Green Fluorescent Protein and observing fluorescence resonance energy transfer (FRET). For example, fusions of CFP, calmodulin, a calmodulin-binding peptide, and YFP are transfectable emission-ratioing Ca2+ indicators with many uses. They are highly suitable for twophoton excitation at 770-810 nm, even at video rates. Applications not possible with previous indicators include detection of submicroscopic domains of Ca2+ by fusion of the indicators to key proteins, and dynamic imaging of Ca2+ in transgenic animals. YFPs have been improved as FRET acceptors by reducing their sensitivity to pH changes. Many other applications of GFP mutants to detect fluctuating protein-protein interactions are underway.

A synthetic alternative to GFPs for protein tagging arises from the ability of membrane-permeant biarsenical dyes to seek out and light up alpha-helical Cys-Cys-X-X-Cys-Cys motifs placed in recombinant proteins in live cells. The new system is much smaller than GFP (6 residues vs. 238), can label internal domains not just N- and C-terminii, and offers novel readouts (e.g. red emission peaking > 600 nm) and better temporal control of the labeling.

Type
Novel Approaches to Microscopy Of Living Cells
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 1036 - 1037
Copyright
Copyright © Microscopy Society of America

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References

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