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We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.
Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.
Protein-protein interactions (PPIs) control various key processes in cells1. The roles of PPIs differ based on protein composition, affinities functions and locations in cells2. PPIs can be investigated via different techniques3. For example, co-immunoprecipitation is a relatively simple, robust, and inexpensive technique commonly used tool to identify or confirm PPIs. However, studying PPIs can be challenging when the interacting proteins have low expression levels or when the interactions are transient or relevant only in specific environments. Studying PPIs occurring between the different enzymes of the pyoverdine pathway in P. aeruginosa requires that the repression of the general iron-co-factored repressor Fur is relieved to allow the expression of all the proteins of the pyoverdine pathway to be expressed in the cell4,5,6. This common regulation for all the proteins of the pathway results in timely expressions in the cell expected to promote their interactions. The diversity in term of size, nature, expression levels and the number of proteins of this metabolic pathway make it difficult for study in reconstituted systems6. Exploring PPIs in their cellular environment is therefore critical to further understand the biological functions of proteins in their native context.
Only few methods including fluorescence allow exploring PPIs in living cells7. Amongst the different fluorescence parameters that can be measured, the fluorescence lifetime (i.e., the average time a fluorophore remains in its excited state before emitting a photon) is likely one of the most interesting parameters to explore in living cells. The fluorescence lifetime of a fluorophore is highly sensitive to its environment and FLIM can therefore provide chemical or physical information regarding the fluorophore surroundings8. This includes the presence of Förster resonance energy transfer (FRET) that can occur in the presence of an “acceptor” of fluorescence located at a short distance of a fluorescence “donor”. Energy transfer results in significant shortening of the donor fluorescence lifetime (Figure 1A), making Fluorescence Lifetime Imaging Microscopy (FLIM) a powerful approach to explore protein-protein interactions directly in live cells. FLIM can additionally provide spatial information about where the interactions take place in cells7,8. This approach is extremely powerful for investigating PPIs in situations where the labeling with fluorophores of the two interacting partners is possible.
For FRET to occur - critical conditions on the distance between two fluorophores are required8,9. The two fluorophores should not be distant from each other by more than 10 nm. Therefore, cautions must be taken when designing FLIM-FRET experiments to ensure that the donor and the acceptor of fluorescence have a chance to be located close to each other in the interacting complex. While this may seem constraining, it is in fact a true advantage as the distance-dependence of FRET ensures that two labelled proteins undergoing FRET have to physically interact (Figure 1A). The difficulties at getting clear answers about PPI in colocalization experiments (two colocalized proteins may not necessarily interact) are therefore not an issue using FLIM-FRET.
Figure 1: FLIM-FRET analysis principle. Each pixel of the FLIM-FRET multidimensional image contains information about the fluorescence decay recorded at this particular location (#counts = number of detected photons in the channel t). (A) The classical representation of the FLIM image is usually a false-color lifetime encoded 2D image (left). A decrease in the mean fluorescence lifetime of the donor - as seen by a change in the color scale - can be observed in the presence of FRET and is informative about the presence of PPIs in this spatial area. (B) Overlap between the donor emission spectrum and the acceptor absorption spectrum is necessary for FRET to occur. Please click here to view a larger version of this figure.
A second requirement for FRET is that the emission spectrum of the donor and the absorption spectra of the acceptor should overlap8 (Figure 1B). The fluorescence excitation of the donor should be at wavelengths that contribute very little to the direct fluorescence excitation of the acceptor. Not all combinations of fluorophores are possible and we additionally recommend to preferentially use donors with monoexponential fluorescence decays to facilitate FLIM-FRET interpretations10. Several couples of fluorescence proteins meet these requirements, including the popular eGFP-mCherry couple11 (for a review on the palette of available fluorescent protein FRET pairs see12,13).
FLIM-FRET allows measuring the fluorescence lifetime decay of a FRET donor at every pixel of a FLIM image (Figure 1A). There are two major techniques to determine fluorescence lifetime that differ in acquisition and analysis: frequency-domain (FD)14 and time-domain (TD). TD FLIM is more widespread and is performed using a pulsed illumination combined with different possible detection configurations including gating methods15, streak camera16 or time-correlated single photon counting (TCSPC) techniques8. For both FD and TD techniques, fluorescence lifetime is not directly measured but requires an analysis of the measured data to estimate the lifetime(s) or the presence of interactions. For TCSPC techniques, the most widely used analysis relies on fitting the decays with single or multi exponential functions using least square iterative re-convolutions that minimize the weighted sum of the residuals.
Finally, FLIM-FRET can be performed both by using single photon or multiphoton excitations. The latest have several advantages like reducing autofluorescence and photodamage out of the focal plane. Multiphoton excitations allow also a longer excitation depth if working in thick 3D samples8. On the contrary, single photon excitation is usually more efficient as the two-photon absorption cross sections of fluorescent proteins are limited17.
Here, we propose a protocol for FLIM-FRET measurements of PPIs in live P. aeruginosa in the particular case of two interacting proteins (PvdA and PvdL) expressed with highly different numbers of copies to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. PvdA and PvdL proteins are involved in pyoverdine biosynthesis. PvdA is a L-ornithine N5-oxygenase and synthesizes the L-N5-formyl-N5-hydroxyornithine from L-ornithine by hydroxylation (PvdA) and formylation (PvdF)18. PvdL is a non-ribosomal peptide synthesis (NRPS) enzyme composed of four modules. The first module catalyzes the acylation of myristic acid. The second module catalyzes the activation of L-Glu and its condensation to the myristic-coA. Then, the third module condenses a L-Tyr amino acid that is then isomerized in D-Tyr. Finally, the fourth module binds a L-Dab (Diaminobutyric acid) amino acid to form the acylated tripeptide L-Glu/D-Tyr/L-Dab6. PvdL is thus responsible for the synthesis of the three first amino acids of the pyoverdine precursor. The interaction of PvdA protein with PvdL is surprising as PvdL, on the contrary to PvdI and PvdJ, does not carry a module specific for the L-N5-formyl-N5-hydroxyornithine. This interaction suggest that all the enzymes responsible for the pyoverdine precursor biosynthesis are arranged in large transient and dynamic multi-enzymatic complexes19,20.
In this report we explain in detail how to construct the bacterial strains expressing natively the two interacting eGFP and mCherry labelled proteins. We also describe sample preparation and conditions for efficient FLIM-FRET cell imaging. Finally, we propose a step-by-step tutorial for image analysis including a recently developed tool providing advanced visualization possibilities for straightforward interpretation of complex FLIM-FRET data. With this report, we would like to convince not only adventurous but most biologists that FRET-FLIM is an accessible and powerful technique able to address their questions about PPIs directly in the native cellular environment.
1. Plasmid construction
Figure 2: Overview of PCR strategy and plasmids construction used for the construction of PvdA-mCherry. See text for details - pvdA encodes an enzyme involved in the biosynthesis of the siderophore pyoverdine, a secondary metabolite involved in iron acquisition. Please click here to view a larger version of this figure.
2. Fluorescent tag insertion into the chromosomal genome of P. aeruginosa (Figure 3)
Figure 3: Protocol of construction of P. aeruginosa strains by fluorescent tag insertion. See text for details. Please click here to view a larger version of this figure.
3. Pyoverdine measurement
4. Bacteria culture and conditions for cells to express PvdA, PvdL and PvdJ
5. Preparation of agarose pad (Figure 4)
Figure 4: Agarose pad preparation. Please click here to view a larger version of this figure.
6. Imaging with a two-photon microscopy setup
NOTE: We are using a home-made two-photon excitation scanning inverted microscope with a 60x 1.2NA water immersion objective operating in de-scanned fluorescence collection mode. Two-photon excitation wavelength is set at 930 nm. It is provided by a Ti:Sapphire laser (80 MHz repetition rate, ≈ 70 fs pulse width) working at 10-20 mW. Fluorescence photons were collected through a 680 nm short pass filter and a 525/50 nm band-pass filter before being directed to a fiber-coupled avalanche photo-diode connected to a time-correlated single photon counting (TCSPC) module. The microscope is also equipped with a transmission fluorescence lamp. Several FLIM-FRET microscopes are now commercially available and many imaging facilities are equipped with setups able to perform FLIM-FRET measurements.
Figure 5: Schematic representation of the interface of microscope control software. Please click here to view a larger version of this figure.
7. Data analysis
Figure 6: Main panel of the data analysis window of SPCImage software. Intensity image (blue box), lifetime image (purple box), lifetime histogram (upper right), decay curve at selected position (green box), and decay parameters at selected position (cyan box) of a representative PvdA-eGFP decay recorded in live P. aeruginosa using a bh SPC830 acquisition card on a home-made Two-Photon Excitation-FLIM-FRET setup. The experimental decay curve of the pixel pointed in the above image, its mono-exponential fit (red curve) deconvoluting the decay from its calculated instrumental response function (green curve) can be seen in the green panel. Please click here to view a larger version of this figure.
Figure 7: (A) Algorithm settings for fitting the decays with exponential models. Selecting MLE (maximum-likelihood algorithm or maximum-likelihood estimation, MLE) as the fit model, and (B) export options window. Please click here to view a larger version of this figure.
Empirical cumulative distribution functions (ecdf) of the fluorescence lifetimes measured for the different bacterial strains are shown in Figure 8. If FRET occurs, the ecdfs are shifted towards the shorter-lived lifetimes (Figure 8A,8B). Note that when the interaction of the two proteins results in a long distance between the two fluorophores, no FRET can occur (Figure 8C). This situation cannot be distinguished fr...
FLIM-FRET offers some key advantages over intensity-based FRET imaging. Fluorescence lifetime is an intrinsic parameter of the fluorophore. As a consequence, it is not dependent on local concentrations of fluorophores neither on the intensity of the light excitation. The fluorescence lifetime is additionally also poorly affected by photo-bleaching. It is particularly interesting to evidence PPIs in cells where local proteins concentrations can be highly heterogeneous throughout the subcellular compartments or regions. FL...
The authors have nothing to disclose.
We acknowledge Dr Ludovic Richert for his valuable assistance on FLIM data acquisition and for the technical maintenance and development of the FLIM setup. This work was funded by grants from Fondation pour la Recherche en Chimie (https://icfrc.fr/). VN is funded by the Fondation pour la Recherche Médicale (FRM‐SPF201809006906). YM is grateful to the Institut Universitaire de France (IUF) for support and providing additional time to be dedicated to research. IJS and JG acknowledge the Institute on Drug Delivery of Strasbourg for its financial support.
Name | Company | Catalog Number | Comments |
525/50 nm band-pass filter | F37-516, AHF, Germany | ||
680 nm short pass filter | F75-680, AHF, Germany | ||
Agarose | Sigma-Aldrich | A9539 | |
Ammonium Sulfate (NH4)2SO4 | Sigma-Aldrich | A4418 | |
DreamTaq DNA polymerase 5U/μL | ThermoFisher Scientific | EP0714 | |
E. coli TOP10 | Invitrogen | C404010 | |
Fiber-coupled avalanche photo-diode | SPCM-AQR-14- FC, Perkin Elmer | ||
Glass coverslips (Thickness No. 1.5, 20×20mm | Knitel glass | MS0011 | |
High-Fidelity DNA polymerase Phusion 2U/μL | ThermoFisher Scientific | F530S | |
Lysogeny broth (LB) | Millipore | 1.10285 | |
Magnesium Sulfate Heptahydrate (MgSO4 . 7H2O) | Sigma-Aldrich | 10034-99-8 | |
Microscope slides (25×75mm) | Knitel glass | MS0057 | |
NucleoSpin Gel and PCR Clean-up | Macherey-Nagel | 740609.50 | |
NucleoSpin Plasmid | Macherey-Nagel | 740588.10 | |
Potassium Phosphate Dibasic (K2HPO4) | Sigma-Aldrich | RES20765 | |
Potassium Phosphate Monobasic (KH2PO4) | Sigma-Aldrich | P5655 | |
Sodium Succinate (Disodium) | Sigma-Aldrich | 14160 | |
SPCImage, SPCM software | Becker & Hickl | ||
Sterile inoculating loop | Nunc | 7648-1PAK | |
T4 DNA ligase 1U/μL | ThermoFisher Scientific | 15224017 | |
TCSPC module | SPC830, Becker & Hickl, Germany | ||
Ti:Sapphire laser | Insight DeepSee, Spectra Physics | ||
Tubes 50mL | Falcon | 352070 |
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