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N-way FRET microscopy of multiple protein-protein interactions in live cells.

Hoppe AD, Scott BL, Welliver TP, Straight SW, Swanson JA - PLoS ONE (2013)

Bottom Line: Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells.We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed.Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, South Dakota State University, Brookings, South Dakota, United States of America. adam.hoppe@sdstate.edu

ABSTRACT
Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

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N-Way FRET recovers concentrations and apparent FRET efficiencies from cells expressing FP-FP fusions.Cells were transfected with CFP-YFP (A and B), RFP-CFP (C and D), RFP-YFP (E and F), RFP-darkFP-CFP (G) and RFP-kinesin-CFP (H). The raw images (top rows, independently scaled) were analyzed by N-Way FRET, using B−1, to produce the concentration estimates (bottom rows) ([FP], display scale, 0–1,400 intensity units) and the apparent FRET efficiencies (EFP-FP[FP-FP], display scale 0–500 intensity units). Images are representative of 20 cells per condition. Plots of concentration estimates (C, D, F, G and H) indicated that N-Way FRET accurately recovered the correct one-to-one stoichiometry of each FP in the sample as well as their apparent efficiencies (n = 20 for each). Note the decreasing FRET efficiency observed for increasing size of inserted peptide or proteins: high FRET (RFP-CFP, D), low FRET (darkFP, G) or no FRET (kinesin, H) seen in ECR[CR]. Data from Scope 1.
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pone-0064760-g003: N-Way FRET recovers concentrations and apparent FRET efficiencies from cells expressing FP-FP fusions.Cells were transfected with CFP-YFP (A and B), RFP-CFP (C and D), RFP-YFP (E and F), RFP-darkFP-CFP (G) and RFP-kinesin-CFP (H). The raw images (top rows, independently scaled) were analyzed by N-Way FRET, using B−1, to produce the concentration estimates (bottom rows) ([FP], display scale, 0–1,400 intensity units) and the apparent FRET efficiencies (EFP-FP[FP-FP], display scale 0–500 intensity units). Images are representative of 20 cells per condition. Plots of concentration estimates (C, D, F, G and H) indicated that N-Way FRET accurately recovered the correct one-to-one stoichiometry of each FP in the sample as well as their apparent efficiencies (n = 20 for each). Note the decreasing FRET efficiency observed for increasing size of inserted peptide or proteins: high FRET (RFP-CFP, D), low FRET (darkFP, G) or no FRET (kinesin, H) seen in ECR[CR]. Data from Scope 1.

Mentions: As expected, B contains negative values corresponding to the subtractive components for FRET-induced donor losses as suggested by the negative topology expected in the 2D spectrum (Fig. 1). Similar results were obtained for B, when pairs of linked FRET constructs were used. The FRET efficiencies of tandem linked constructs CFP-YFP, CFP-RFP and RFP-YFP were determined by both fluorescence lifetime and acceptor photobleaching (Fig. S6). Here, B was then computed by finding the common γ values (see theory). Both methods for determining B produced similar results (compare Fig. 3, Scope 1, with all other figures). Furthermore, good agreement was observed for FRET efficiency determination by acceptor photobleaching and by fluorescence lifetime (Fig. S6), indicating that either method can be used equivalently for calibration of test constructs for N-Way FRET.


N-way FRET microscopy of multiple protein-protein interactions in live cells.

Hoppe AD, Scott BL, Welliver TP, Straight SW, Swanson JA - PLoS ONE (2013)

N-Way FRET recovers concentrations and apparent FRET efficiencies from cells expressing FP-FP fusions.Cells were transfected with CFP-YFP (A and B), RFP-CFP (C and D), RFP-YFP (E and F), RFP-darkFP-CFP (G) and RFP-kinesin-CFP (H). The raw images (top rows, independently scaled) were analyzed by N-Way FRET, using B−1, to produce the concentration estimates (bottom rows) ([FP], display scale, 0–1,400 intensity units) and the apparent FRET efficiencies (EFP-FP[FP-FP], display scale 0–500 intensity units). Images are representative of 20 cells per condition. Plots of concentration estimates (C, D, F, G and H) indicated that N-Way FRET accurately recovered the correct one-to-one stoichiometry of each FP in the sample as well as their apparent efficiencies (n = 20 for each). Note the decreasing FRET efficiency observed for increasing size of inserted peptide or proteins: high FRET (RFP-CFP, D), low FRET (darkFP, G) or no FRET (kinesin, H) seen in ECR[CR]. Data from Scope 1.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675202&req=5

pone-0064760-g003: N-Way FRET recovers concentrations and apparent FRET efficiencies from cells expressing FP-FP fusions.Cells were transfected with CFP-YFP (A and B), RFP-CFP (C and D), RFP-YFP (E and F), RFP-darkFP-CFP (G) and RFP-kinesin-CFP (H). The raw images (top rows, independently scaled) were analyzed by N-Way FRET, using B−1, to produce the concentration estimates (bottom rows) ([FP], display scale, 0–1,400 intensity units) and the apparent FRET efficiencies (EFP-FP[FP-FP], display scale 0–500 intensity units). Images are representative of 20 cells per condition. Plots of concentration estimates (C, D, F, G and H) indicated that N-Way FRET accurately recovered the correct one-to-one stoichiometry of each FP in the sample as well as their apparent efficiencies (n = 20 for each). Note the decreasing FRET efficiency observed for increasing size of inserted peptide or proteins: high FRET (RFP-CFP, D), low FRET (darkFP, G) or no FRET (kinesin, H) seen in ECR[CR]. Data from Scope 1.
Mentions: As expected, B contains negative values corresponding to the subtractive components for FRET-induced donor losses as suggested by the negative topology expected in the 2D spectrum (Fig. 1). Similar results were obtained for B, when pairs of linked FRET constructs were used. The FRET efficiencies of tandem linked constructs CFP-YFP, CFP-RFP and RFP-YFP were determined by both fluorescence lifetime and acceptor photobleaching (Fig. S6). Here, B was then computed by finding the common γ values (see theory). Both methods for determining B produced similar results (compare Fig. 3, Scope 1, with all other figures). Furthermore, good agreement was observed for FRET efficiency determination by acceptor photobleaching and by fluorescence lifetime (Fig. S6), indicating that either method can be used equivalently for calibration of test constructs for N-Way FRET.

Bottom Line: Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells.We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed.Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, South Dakota State University, Brookings, South Dakota, United States of America. adam.hoppe@sdstate.edu

ABSTRACT
Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

Show MeSH
Related in: MedlinePlus