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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 the relative concentrations and apparent FRET efficiencies from cells expressing FP-FP-FP fusions.Cells were transfected with CFP-RFP-YFP (A and B) and YFP-CFP-RFP (C and D) linked constructs. Spectral FRET data (top row) were unmixed by N-Way FRET to produce the concentration estimates (bottom rows) of the total FP (bound and free, [FP]) and the apparent complex concentrations (EFP-FP[FP-FP]). Plots of concentration estimates (C and D) averaged over 20 cells indicate that N-Way FRET recovered correct one-to-one stoichiometry of all three FPs in the sample and that the FPs on the ends of the polypeptide gave the lowest FRET efficiencies (error bars are standard deviation, 20 cells for each, from Scope 2).
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pone-0064760-g004: N-Way FRET recovers the relative concentrations and apparent FRET efficiencies from cells expressing FP-FP-FP fusions.Cells were transfected with CFP-RFP-YFP (A and B) and YFP-CFP-RFP (C and D) linked constructs. Spectral FRET data (top row) were unmixed by N-Way FRET to produce the concentration estimates (bottom rows) of the total FP (bound and free, [FP]) and the apparent complex concentrations (EFP-FP[FP-FP]). Plots of concentration estimates (C and D) averaged over 20 cells indicate that N-Way FRET recovered correct one-to-one stoichiometry of all three FPs in the sample and that the FPs on the ends of the polypeptide gave the lowest FRET efficiencies (error bars are standard deviation, 20 cells for each, from Scope 2).

Mentions: To examine the ability of N-Way FRET to recover concentrations and efficiencies from samples containing three fluorophores, we generated FP FRET trio constructs (C-R-Y, C-Y-R). While the amount of FRET in these cells is not apparent in the raw data, N-Way FRET analysis revealed FRET between all FPs and returned the expected equimolar concentrations of each FP and FRET signals that scaled with the spacing between FPs (Fig. 4). Cells expressing C-R-Y demonstrated the highest FRET efficiency between C-R, and Y-R, and lower FRET efficiency for C-Y, consistent with the larger distance between C and Y (Fig. 4). In the C-Y-R construct, the lowest FRET efficiency was between the terminal FPs (C and R), as expected (Fig. 4). While the potential for energy migration from C->Y->R exists, N-Way FRET (or any other spectral method) cannot distinguish this from direct transfer C->R. The low FRET efficiency observed for the terminal FPs in both the C-R-Y and C-Y-R constructs suggests energy migration does not contribute substantially. This observation was supported by C-darkFP-R having nearly the same value for E[CR] as in C-Y-R indicating that Y was not significantly facilitating transfer of energy from C to R (Fig. 3 and 4). Together, these data indicate that N-Way FRET can accurately recover the abundances and apparent FRET efficiencies for model constructs.


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 the relative concentrations and apparent FRET efficiencies from cells expressing FP-FP-FP fusions.Cells were transfected with CFP-RFP-YFP (A and B) and YFP-CFP-RFP (C and D) linked constructs. Spectral FRET data (top row) were unmixed by N-Way FRET to produce the concentration estimates (bottom rows) of the total FP (bound and free, [FP]) and the apparent complex concentrations (EFP-FP[FP-FP]). Plots of concentration estimates (C and D) averaged over 20 cells indicate that N-Way FRET recovered correct one-to-one stoichiometry of all three FPs in the sample and that the FPs on the ends of the polypeptide gave the lowest FRET efficiencies (error bars are standard deviation, 20 cells for each, from Scope 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675202&req=5

pone-0064760-g004: N-Way FRET recovers the relative concentrations and apparent FRET efficiencies from cells expressing FP-FP-FP fusions.Cells were transfected with CFP-RFP-YFP (A and B) and YFP-CFP-RFP (C and D) linked constructs. Spectral FRET data (top row) were unmixed by N-Way FRET to produce the concentration estimates (bottom rows) of the total FP (bound and free, [FP]) and the apparent complex concentrations (EFP-FP[FP-FP]). Plots of concentration estimates (C and D) averaged over 20 cells indicate that N-Way FRET recovered correct one-to-one stoichiometry of all three FPs in the sample and that the FPs on the ends of the polypeptide gave the lowest FRET efficiencies (error bars are standard deviation, 20 cells for each, from Scope 2).
Mentions: To examine the ability of N-Way FRET to recover concentrations and efficiencies from samples containing three fluorophores, we generated FP FRET trio constructs (C-R-Y, C-Y-R). While the amount of FRET in these cells is not apparent in the raw data, N-Way FRET analysis revealed FRET between all FPs and returned the expected equimolar concentrations of each FP and FRET signals that scaled with the spacing between FPs (Fig. 4). Cells expressing C-R-Y demonstrated the highest FRET efficiency between C-R, and Y-R, and lower FRET efficiency for C-Y, consistent with the larger distance between C and Y (Fig. 4). In the C-Y-R construct, the lowest FRET efficiency was between the terminal FPs (C and R), as expected (Fig. 4). While the potential for energy migration from C->Y->R exists, N-Way FRET (or any other spectral method) cannot distinguish this from direct transfer C->R. The low FRET efficiency observed for the terminal FPs in both the C-R-Y and C-Y-R constructs suggests energy migration does not contribute substantially. This observation was supported by C-darkFP-R having nearly the same value for E[CR] as in C-Y-R indicating that Y was not significantly facilitating transfer of energy from C to R (Fig. 3 and 4). Together, these data indicate that N-Way FRET can accurately recover the abundances and apparent FRET efficiencies for model constructs.

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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