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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    • "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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    ABSTRACT: Genetically encoded fluorescent biosensors have long proven to be excellent tools for quantitative live imaging, but sensor applications in plants have been lacking behind those in mammalian systems with respect to the variety of sensors and tissue types used. How can this be improved, and what can be expected for the use of genetically encoded fluorescent biosensors in plants in the future? In this review, we present a table of successful physiological experiments in plant tissue using fluorescent biosensors, and draw some conclusions about the specific challenges plant cell biologists are faced with and some of the ways they have been overcome so far.
    Frontiers in Plant Science 07/2013; 4:234. DOI:10.3389/fpls.2013.00234 · 3.95 Impact Factor
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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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    ABSTRACT: Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn(2+)based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn(2+) is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn(2+) levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn(2+). Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn(2+) uptake simultaneously in two compartments, revealing that nuclear Zn(2+) rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn(2+) more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn(2+) and reveal that nuclear Zn(2+) is buffered at a higher level than cytosolic Zn(2+).
    PLoS ONE 11/2012; 7(11):e49371. DOI:10.1371/journal.pone.0049371 · 3.23 Impact Factor
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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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    ABSTRACT: Quantitative imaging in live cells is a powerful method for monitoring the dynamics of biomolecules at an excellent spatio-temporal resolution. Such an approach, initially limited to a small number of substrates for which specific dyes were available, has become possible for a large number of biomolecules due to the development of genetically encoded, protein-based sensors. These sensors, which can be introduced into live cells through a transgenic approach, offer the benefits of quantitative imaging, with an extra advantage of non-invasiveness. In the past decade there has been a drastic expansion in the number of biomolecules for which genetically encoded sensors are available, and the functional properties of existing sensors are being improved at a dramatic pace. A number of technical improvements have now made the application of genetically encoded sensors in plants rather straightforward, and some of the sensors such as calcium indicator proteins have become standard analytical tools in many plant laboratories. The use of a handful of probes has already revealed an amazing specificity of cellular biomolecule dynamics in plants, which leads us to believe that there are many more discoveries to be made using genetically encoded sensors. In this short review, we will summarize the progress made in the past 15 years in the development in genetically encoded sensors, and highlight significant discoveries made in plant biology.
    The Plant Journal 04/2012; 70(1):108-17. DOI:10.1111/j.1365-313X.2012.04910.x · 5.97 Impact Factor
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