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Summary

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.

Abstract

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.

Introduction

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 enzym....

Protocol

1. Plasmid construction

  1. Amplify by two PCR (PCR1 and 3) the DNA sequences (use genomic DNA of P. aeruginosa PAO1) of the 700 base pairs upstream and downstream of the regions corresponding to the insertion site in P. aeruginosa genome with high-fidelity DNA polymerase. Add restriction sites to primers in blue and green and add an overlapping sequence with mCherry to primers in red (Figure 2).
    1. For PvdA labelled at the C-terminus with eGFP, amplify the.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

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Materials

NameCompanyCatalog NumberComments
525/50 nm band-pass filterF37-516, AHF, Germany
680 nm short pass filterF75-680, AHF, Germany
AgaroseSigma-AldrichA9539
Ammonium Sulfate (NH4)2SO4Sigma-AldrichA4418
DreamTaq DNA polymerase 5U/μLThermoFisher ScientificEP0714
E. coli TOP10InvitrogenC404010
Fiber-coupled avalanche photo-diodeSPCM-AQR-14- FC, Perkin Elmer
Glass coverslips (Thickness No. 1.5, 20×20mmKnitel glassMS0011
High-Fidelity DNA polymerase Phusion 2U/μLThermoFisher ScientificF530S
Lysogeny broth (LB)Millipore1.10285
Magnesium Sulfate Heptahydrate (MgSO4 . 7H2O)Sigma-Aldrich10034-99-8
Microscope slides (25×75mm)Knitel glassMS0057
NucleoSpin Gel and PCR Clean-upMacherey-Nagel740609.50
NucleoSpin PlasmidMacherey-Nagel740588.10
Potassium Phosphate Dibasic (K2HPO4)Sigma-AldrichRES20765
Potassium Phosphate Monobasic (KH2PO4)Sigma-AldrichP5655
Sodium Succinate (Disodium)Sigma-Aldrich14160
SPCImage, SPCM softwareBecker & Hickl
Sterile inoculating loopNunc7648-1PAK
T4 DNA ligase 1U/μLThermoFisher Scientific15224017
TCSPC moduleSPC830, Becker & Hickl, Germany
Ti:Sapphire laserInsight DeepSee, Spectra Physics
Tubes 50mLFalcon352070

References

  1. Braun, P., Gingras, A. C. History of protein-protein interactions: From egg-white to complex networks. Proteomics. 12, 1478-1498 (2012).
  2. Nooren, I. M. A., Thornton, J. M. Structur....

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