scholarly journals Förster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells

2013 ◽  
Vol 83 (9) ◽  
pp. 780-793 ◽  
Author(s):  
Yuansheng Sun ◽  
Christina Rombola ◽  
Vinod Jyothikumar ◽  
Ammasi Periasamy
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Förster Resonance

scholarly journals Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

2008 ◽  
Vol 363 (1500) ◽  
pp. 2143-2151 ◽  
Author(s):  
Kazuhiro Aoki ◽  
Etsuko Kiyokawa ◽  
Takeshi Nakamura ◽  
Michiyuki Matsuda
Keyword(s):  
Signal Transduction ◽  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Small Gtpases ◽  
Forster Resonance Energy Transfer ◽  
Fluorescence Probes ◽  
Förster Resonance Energy Transfer ◽  
Förster Resonance

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine–threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

scholarly journals Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Anca Margineanu ◽  
Jia Jia Chan ◽  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Delphine Flatters ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Förster Resonance

Abstract We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. K D values broadly agree with published biochemical measurements.

scholarly journals A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission

2008 ◽  
Vol 6 (suppl_1) ◽  
Author(s):  
A.D Elder ◽  
A Domin ◽  
G.S Kaminski Schierle ◽  
C Lindon ◽  
J Pines ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Confocal Imaging ◽  
Förster Resonance Energy Transfer ◽  
Förster Resonance

Fluorescence detection of acceptor molecules sensitized by Förster resonance energy transfer (FRET) is a powerful method to study protein interactions in living cells. The method requires correction for donor spectral bleed-through and acceptor cross-excitation as well as the correct normalization of signals to account for varying fluorophore concentrations and imaging parameters. In this paper, we review different methods for FRET signal normalization and then present a rigorous model for sensitized emission measurements, which is both intuitive to understand and practical to apply. The method is validated by comparison with the acceptor photobleaching and donor lifetime-imaging techniques in live cell samples containing EYFP and ECFP tandem constructs exhibiting known amounts of FRET. By varying the stoichiometry of interaction in a controlled fashion, we show that information on the fractions of interacting donors and acceptors can be recovered. Furthermore, the method is tested by performing measurements on different microscopy platforms in both widefield and confocal imaging modes to show that signals recovered under different imaging conditions are in quantitative agreement. Finally, the method is applied in the study of dynamic interactions in the cyclin–cdk family of proteins in live cells. By normalizing the obtained signals for both acceptor and donor concentrations and using a FRET exhibiting control construct for calibration, stoichiometric changes in these interactions could be visualized in real time. The paper is written to be of practical use to researchers interested in performing sensitized emission measurements. The correct interpretation of the retrieved signals in a biological context is emphasized, and guidelines are given for the practical application of the developed algorithms.

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