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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 enzym....
1. Plasmid construction
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.......
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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