Improving the spectral analysis of fluorescence resonance energy transfer in live cells: Application to interferon receptors and Janus kinases

•Developed protocol to accurately measure FRET between two proteins in live cells.•Ratio and levels of acceptor and donor affected observed FRET efficiency.•Using blue-shifted fluorophores erroneously increased the observed FRET efficiency.•The affinity and specificity of cellular interactions can b...

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Bibliographic Details
Published in:Cytokine (Philadelphia, Pa.) Pa.), 2013-10, Vol.64 (1), p.272-285
Main Authors: Krause, Christopher D., Digioia, Gina, Izotova, Lara S., Pestka, Sidney
Format: Article
Language:English
Subjects:
Animals
Cell Line
Chlorocebus aethiops
COS Cells
cytokines
energy transfer
Equilibrium
fluorescence
Fluorescence Resonance Energy Transfer - methods
Fluorescent Dyes - metabolism
FRET
Green Fluorescent Proteins - metabolism
HEK293 Cells
Humans
Interferon
Interferon gamma Receptor
Interferon-gamma - metabolism
interferons
Janus kinase
Janus Kinases - analysis
kinases
Luminescent Proteins - metabolism
Microscopy, Fluorescence - methods
Multiprotein Complexes
Receptor
receptors
Receptors, Interferon - analysis
Receptors, Interferon - metabolism
Red Fluorescent Protein
Scyphozoa
spectral analysis
Staining and Labeling
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Summary:•Developed protocol to accurately measure FRET between two proteins in live cells.•Ratio and levels of acceptor and donor affected observed FRET efficiency.•Using blue-shifted fluorophores erroneously increased the observed FRET efficiency.•The affinity and specificity of cellular interactions can be compared.•The ability of cellular interactions to achieve equilibrium can be judged. The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
ISSN:1043-4666
1096-0023
DOI:10.1016/j.cyto.2013.05.026