FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

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 700 bp region upstream relative to the stop codon by the primers in blue, and amplify the 700 bp downstream region containing the stop codon with the....

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.......

Materials

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