Multiplexed FRET to Image Multiple Signaling Events in Live Cells

Chemical Biology Centre, Imperial College London, United Kingdom.
Biophysical Journal (Impact Factor: 3.97). 09/2008; 95(10):L69-71. DOI: 10.1529/biophysj.108.139204
Source: PubMed


We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.

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Available from: Ewan J Mcghee
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    • "Expanding the variety of sensor fluorescent proteins, e.g., by the development of different FRET donor/acceptors would facilitate the study of several ions/metabolites simultaneously, e.g., the commonly linked signaling cascade of intracellular calcium/apoplastic pH, as well as same ion fluxes in several compartments or complex protein-protein interactions. Multiplexed FRET (Piljic and Schultz, 2008) and fluorescence lifetime imaging (FLIM)-FRET (Grant et al., 2008) are becoming more feasible as the variety of spectral variants increases. In N. benthamiana leaves a FRET-FLIM assay was used to detect known protein interactions using a FRET pair of the GFP variant TSapphire as donor, and mOrange as acceptor (Bayle et al., 2008). "
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    • "Secondly, the biophysical (folding, maturation, oligomerization state) and photophysical properties (brightness) of red and orange FPs still lag behind those of the cyan-yellow counterparts [9], making it challenging to identify a robust alternate FRET pair. Indeed of the non-CFP/YFP biosensors developed thus far, each research team chose a different combination of FRET partners [5], [10], [11], [12], [13], [14]. "
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    • "Dual imaging using multiple genetically encoded sensors will be beneficial to resolve the temporal correlation between two interdependent signaling molecules, similar to the case of calcium and ROS described above. Spectrally orthogonal FRET pairs such as eCFP/eYFP and mOrange/ mCherry (Piljic and Schultz, 2008), mTFP/Citrine and mAmetrine/tdTomato (Ai et al., 2008), eCFP/mVenus and TagRFP/ mPlum (Grant et al., 2008), and CFP/YFP and Sapphire/RFP (Niino et al., 2009) have recently been reported. In addition, the development of single-FP sensors would alleviate the problem of spectral overlapping and hence make dualimaging more feasible. "
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