Application of phasor plot and autofluorescence correction for study of heterogeneous cell population

Abstract

Abstract. Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.

A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza

“…2R9 binding to TLR1 and TLR6 was weaker than that for two other TLR2 peptides (Figure S5A–B); however, we noted considerable binding of 2R9 to TLR4 (Figure S5C). 2R9/TLR4 binding was only slightly less than that of 4BB, a TLR4-derived peptide that strongly binds TLR4 (Szmacinski et al, 2014; Toshchakov et al, 2011). Interestingly, regions of TIR surface represented by 2R1 and 2R3 peptides are juxtaposed (Figure S1B) and both peptides preferentially interact with TLR2 co-receptors, TLR1 and TLR6.…”

“…Cerulean- (Cer-) labeled TIR domains and Bodipy-TMR-X- (BTX-) labeled decoy peptides were used as a FRET pair to study interactions of decoy peptides with TIR domains (Toshchakov et al, 2011). We recently developed a Fluorescence Lifetime IMaging- (FLIM-) based approach that enables quantitative comparisons of TIR-peptide binding affinities directly in cells (Szmacinski et al, 2014). This approach was used to analyze new FLIM images.…”

“…Therefore, TLR1, MyD88, and TIRAP TIR domains were selected as the primary candidate targets of TLR2-derived decoy peptides, and mammalian expression vectors that encode these TIR domains fused at the C terminus with Cer were generated. Figures 3A–C present results of analysis of Cer fluorescence lifetime in images of HeLa cells transfected with Cer-fused, TIR-containing proteins and incubated in the presence of different concentrations of BTX-labeled 2R9 (BTX-2R9) for 1 h. Images were analyzed using the bi-exponential fluorescence decay model with the fixed components’ lifetimes of 2.96 ns (free donor) and 0.9 ns (donor-acceptor pair), determined as described previously (Szmacinski et al, 2014). Figure 3A demonstrates cell images in pseudocolor based on the fractional amplitude of the short lifetime component, α 1 ; this parameter reflects the portion of acceptor-bound donor molecules (Szmacinski et al, 2014).…”

“…Figures 3A–C present results of analysis of Cer fluorescence lifetime in images of HeLa cells transfected with Cer-fused, TIR-containing proteins and incubated in the presence of different concentrations of BTX-labeled 2R9 (BTX-2R9) for 1 h. Images were analyzed using the bi-exponential fluorescence decay model with the fixed components’ lifetimes of 2.96 ns (free donor) and 0.9 ns (donor-acceptor pair), determined as described previously (Szmacinski et al, 2014). Figure 3A demonstrates cell images in pseudocolor based on the fractional amplitude of the short lifetime component, α 1 ; this parameter reflects the portion of acceptor-bound donor molecules (Szmacinski et al, 2014). Incubation of TIRAP-Cer-expressing cells with BTX-2R9 caused a significant, dose-dependent increase of α 1 , reflecting the higher molecular fraction of donor-acceptor pairs.…”

Phasor plotting with frequency-domain flow cytometry

“…The fluorescence lifetime imaging microscopy (FLIM) and fluorimetry communities have been implementing phasor plots for several years [19,[23][24][25][26][27] because of the ease with which one can visualize distributions of multiple lifetime components taken from the different pixels comprising images of groups of cells. The use of phasor plots can aid evaluation of intracellular release of therapeutics from nanoparticles that are taken up by cells [20], detect changes in the metabolism (i.e.…”