Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Summary

Förster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

Abstract

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

Introduction

Microscopy-based analysis of Förster Resonance Energy Transfer (FRET) permits assessment of interactions between proteins in live cells. It provides spatial and temporal information, including information on where in the cell and in which subcellular compartment the interaction takes place and if this interaction changes over time.

Theodor Förster laid the theoretical foundation of FRET in 1948¹. FRET is a radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance of the molecules and the relative orientation of their transition dipoles as well as the overla....

Protocol

1. Plasmid construction

1. For generating the eGFP-mCherry1 fusion probe, use an N1 mammalian cell expression vector (see Table of Materials) with mCherry1¹⁰ inserted using the restriction sites AgeI and BsrGI.
2. Use the following oligonucleotides to amplify eGFP¹¹ without a stop codon as SalI-BamHI fragment: N-terminal primer 5'-AAT TAA CAG TCG ACG ATG GTG AGC AAG GGC GAG G 3' and C-terminal primer 5'-AAT ATA TGG ATC CCG CTT GTA CAG CTC GTC CAT GC 3'.
3. Insert this SalI-BamHI fragment into the multiple cloning site of the N1 ve....

Results

Figure 1 shows the images obtained in the donor channel, channel 1 (488, 505-530 nm), the transfer channel, channel 2 (488, >585 nm), and the acceptor channel, channel 3 (561, >585 nm), respectively. Representative images of cells expressing GFP only, Cherry only, co-expressing GFP and Cherry, and expressing the GFP-Cherry fusion protein. The mean cellular FRET efficiencies calculated in NRK cells expressing GFP-Cherry fusion protein (positive co.......

Discussion

The presented protocol details the use of the genetically coupled one-to-one fluorescent protein calibration probe for quantifying FRET using the detection of sensitized emission of the acceptor and quenching of the donor molecule by confocal microscopy. This method can be applied to assess protein interactions in the physiological context of the living cell in different subcellular compartments. Spatial resolution can be further improved by applying the presented algorithm to calculate FRET efficiencies in each pixel of.......

Materials

Name	Company	Catalog Number	Comments
0.5% Trypsin-EDTA without phenol red (10x)	Thermo Fisher Scientific	15400054
Clontech mCherry N1 vector	Addgene	3553
DMEM without phenol red	Thermo Fisher Scientific	11054020
Fugene 6	Promega	E2691
HEPES	Thermo Fisher Scientific	15630080
LabTek 8-well chambers #1.0	Thermo Fisher Scientific	12565470
L-Glutamine (200 mM)	Thermo Fisher Scientific	25030081