Practical and reliable FRET/FLIM pair of fluorescent proteins

Shcherbo, Dmitry; Souslova, Ekaterina A.; Goedhart, Joachim; Chepurnykh, T. V.; Gaintzeva, Anna; Shemiakina, Irina I.; Gadella, Theodorus W. J.; Lukyanov, Sergey; Chudakov, Dmitriy M.

doi:10.1186/1472-6750-9-24

Cited by 95 publications

(66 citation statements)

References 20 publications

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“…According to our results, when using 820 and 900 nm two-photon excitations, respectively, three possible FRET pair candidates such as the CFP/YFP, YFP/mCherry, and GFP/mCherry showed low acceptor SBT. The GFP/mCherry pair was excluded as a FRET pair based on its lower energy transfer efficiency due to the lower spectral overlap between GFP emission and mCherry (22) but as well to the lower two-photon emission signal compared to YFP upon 900 nm excitation. To evaluate the FRET efficiency of the CFP/YFP and the YFP/mCherry FRET pairs, we next expressed the CFP-YFP and mCherry-YFP tandems in HEK-293 and measured FRET using two-photon FLIM.…”

Section: Screening For a Fret Pair Free From Acceptor Bleedthroughmentioning

confidence: 99%

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim

Kang

et al. 2011

Cytometry Pt A

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Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/ mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells. ' International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; two-photon spectral imaging; unmixing; protein-protein interaction; YFP; mCherry THE current advances in light microscopy and engineering of fluorescent proteins (FPs) with improved properties and altered colors have provided the cellular biologist with the ability to investigate fine molecular events in living cells (1,2). Properly combined, fluorescent molecules allow the study of protein conformational changes or inter protein interaction by means of fluorescence resonance energy transfer (FRET) (3,4). When an excited donor fluorophore is proximate (=10 nm) and suitably oriented to an acceptor molecule, FRET occurs through a nonradiative dipoledipole interaction. Currently, the most popular FP pairs for FRET analysis are cyan with yellow fluorescent protein (CFP/YFP) and green with red fluorescent protein (GFP/RFP) (5,6).Among the methodologies based on nonradiative resonance energy transfer, two approaches prevail, the fluorescence lifetime imaging microscopy (FLIM), and the fluorescence intensity-based FRET imaging. The FLIM is known to be cumbersome to require highly specialized equipment and as well to involve long acquisition time (7). To the contrary, fluorescence intensity-based FRET estimation provides

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Section: Screening For a Fret Pair Free From Acceptor Bleedthroughmentioning

confidence: 99%

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Kim

Kang

et al. 2011

Cytometry Pt A

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“…By fusing a FRET pair using a peptide linker that contains recognition and cleavage sites, proteolysis can be detected in the living cell [71][72][73][74]. For example, FRET-based GEIs containing the DEVD caspase cleavage sequence have been used to monitor apoptosis [69]. In the absence of caspase activity, the FPs remain covalently attached, and their close proximity results in a high FRET state.…”

Section: Conclusion and Future Perspectivementioning

confidence: 99%

“…If the acceptor is in close proximity, the lifetime is reduced. The reduction of the fluorescence lifetime is a kinetic parameter that can be determined independently of the probe concentration, microscope optical path and moderate levels of photobleaching [69].…”

Section: Reliability Reproducibility and Sensitivity Of The Assaysmentioning

confidence: 99%

Nanoparticle-based Caspase Sensors

Maysinger

Hutter

2015

Nanomedicine (Lond.)

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Recent advances in nanotechnology have provided new tools for measuring enzymatic activities that are relevant for the assessment of physiological and pathological processes. Caspases, the enzymes intimately linked with cell death and inflammation, are cysteine-dependent aspartate-directed proteases. The measurement of caspase activity requires assays that can provide data with specificity, precision and sensitivity. Several nanoparticle-based assays are now beginning to emerge. This article will first provide a brief discussion of conventional methods of measuring caspase activity and their limitations, followed by an overview of the advantages and limitations of nanoparticle-based strategies for sensing caspase enzymatic activity in vitro and in vivo.

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“…2). These and related questions can be addressed in the future with the SCG explant migration assay using in vivo reporters, e.g., for proliferation or cell death [32][33][34] in combination with time-lapse imaging. Model of "graded" NRG1-ErbB2/3 signaling in developing Schwann cells.…”

Section: Perspectivesmentioning

confidence: 99%