Quantitative FRET measurement based on spectral unmixing of donor,acceptor and spontaneous excitation‐emission spectra

The spontaneous excitation‐emission (ExEm) spectrum is introduced to the quantitative mExEm‐spFRET methodology we recently developed as a spectral unmixing component for quantitative fluorescence resonance energy transfer measurement, named as SPEES‐FRET method. The spectral fingerprints of both donor and acceptor were measured in HepG2 cells with low autofluorescence separately expressing donor and acceptor, and the spontaneous spectral fingerprint of HEK293 cells with strong autofluoresence was measured from blank cells. SPEES‐FRET was performed on improved spectrometer‐microscope system to measure the FRET efficiency (E) and concentration ratio (R _C) of acceptor to donor vales of FRET tandem plasmids in HEK293 cells, and obtained stable and consistent results with the expected values. Moreover, SPEES‐FRET always obtained stable results for the bright and dim cells coexpressing Cerulean and Venus or Cyan Fluorescent Protein (CFP)‐Bax and Yellow fluorescent protein (YFP)‐Bax, and the E values between CFP‐Bax and YFP‐Bax were 0.02 for healthy cells and 0.14 for the staurosporine (STS)‐treated apoptotic cells. Collectively, SPEES‐FRET has very strong robustness against cellular autofluorescence, and thus is applicable to quantitative evaluation on the protein‐protein interaction in living cells with strong autofluoresence.      

A Method to Quantify FRET Stoichiometry with Phasor Plot Analysis and Acceptor Lifetime Ingrowth

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (K_d) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.     

FRET Spectrometry: A New Tool for the Determination of Protein Quaternary Structure in Living Cells

Förster resonance energy transfer (FRET) is an exquisitely sensitive method for detection of molecular interactions and conformational changes in living cells. The recent advent of fluorescence imaging technology with single-molecule (or molecular-complex) sensitivity, together with refinements in the kinetic theory of FRET, provide the necessary tool kits for determining the stoichiometry and relative disposition of the protomers within protein complexes (i.e., quaternary structure) of membrane receptors and transporters in living cells. In contrast to standard average-based methods, this method relies on the analysis of distributions of apparent FRET efficiencies, E_app, across the image pixels of individual cells expressing proteins of interest. The most probable quaternary structure of the complex is identified from the number of peaks in the E_app distribution and their dependence on a single parameter, termed pairwise FRET efficiency. Such peaks collectively create a unique FRET spectrum corresponding to each oligomeric configuration of the protein. Therefore, FRET could quite literally become a spectrometric method—akin to that of mass spectrometry—for sorting protein complexes according to their size and shape.     

Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells

Quantitative analysis in Förster resonance energy transfer (FRET) experiments in live cells for protein interaction studies is still a challenging issue. In a two-component system (FRET and no FRET donor species), fitting of fluorescence lifetime imaging microscopy (FLIM) data gives the fraction of donor molecules involved in FRET (f_D) and the intrinsic transfer efficiency. But when fast FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are not reliable, even for single lifetime donors. We introduce the new concept of a minimal fraction of donor molecules involved in FRET (mf_D), coming from the mathematical minimization of f_D. We find particular advantage in the use of mf_D because it can be obtained without fitting procedures and it is derived directly from FLIM data. mf_D constitutes an interesting quantitative parameter for live cell studies because it is related to the minimal relative concentration of interacting proteins. For multi-lifetime donors, the process of fitting complex fluorescence decays to find at least four reliable lifetimes is a near impossible task. Here, mf_D extension for multi-lifetime donors is the only quantitative determinant. We applied this methodology for imaging the interaction between the bromodomains of TAF_II250 and acetylated histones H4 in living cells at high resolution. We show the existence of discrete acetylated chromatin domains where the minimal fraction of bromodomain interacting with acetylated H4 oscillates from 0.26 to 0.36 and whose size is smaller than half of one micron cube. We demonstrate that mf_D by itself is a useful tool to investigate quantitatively protein interactions in live cells, especially when using fast FRET-FLIM acquisition times.     

Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f_D) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f_D percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f_D value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f_D (mf_D) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f_D analysis.     

Implementation of Transportation Distance for Analyzing FLIM and FRET Experiments

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.     

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M₂ muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M₂ receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M₂ receptor to enhanced green fluorescent protein-M₂ receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 ± 0.019; n = 4, 0.202 ± 0.010; n = 6, 0.128 ± 0.006; n = 8, 0.093 ± 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20–0.24, identifying the M₂ receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.     

Ab Initio Derivation of the FRET Equations Resolves Old Puzzles and Suggests Measurement Strategies

Quantitative imaging methods based on Förster resonance energy transfer (FRET) rely on the determination of an apparent FRET efficiency (E_app), as well as donor and acceptor concentrations, to uncover the identity and relative abundance of the supramolecular (or quaternary) structures of associating macromolecules. Theoretical work has provided “structure-based” relationships between E_app distributions and the quaternary structure models that underlie them. By contrast, the body of work that predicates the “signal-based” dependence of E_app on directly measurable quantities (i.e., fluorescence emission of donors and acceptors) relies largely on plausibility arguments, one of which is the seemingly obvious assumption that the fraction of fluorescent molecules in the ground state pretty nearly equals the total concentration of molecules. In this work, we use the kinetic models of fluorescence in the presence and absence of FRET to rigorously derive useful relationships between E_app and measurable fluorescence signals. Analysis of these relationships reveals a few anticipated results and some unexpected explanations for known experimental FRET puzzles, and it provides theoretical foundations for optimizing measurement strategies.     

Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).     

Single Cell FRET Analysis for the Identification of Optimal FRET-Pairs in Bacillus subtilis Using a Prototype MEM-FLIM System

Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.     

In vivo FLIM-FRET measurements of recombinant proteins expressed in filamentous fungi

Fluorescence imaging techniques are extremely powerful tools in cell biology, providing valuable insights into the structure and function of biomolecules in their native environments. In particular, the use of Förster resonance energy transfer (FRET) has become increasingly important to obtain information on interactions on the nanoscale, in turn providing insights into molecular behaviour inside living cells. This review describes the basic principles of FRET and fluorescence lifetime imaging microscopy (FLIM) and their application in analyses of protein interactions inside living fungal cells.