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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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Related in: MedlinePlus

N-Way FRET on the excitation-emission landscape.Spectroscopically, FRET is a coupling between donor excitation and acceptor emission. This excitation-emission coupling (Φ) can be described by the outer product of the excitation vector ε and an emission vector s. The Φ signatures define the spectral library A for the N-Way FRET linear unmixing problem (d = Ax = Bc) that can be viewed on the 2D excitation-emission landscape in addition to viewing the data (d). Specifically, these appear as topographical features with light green = 0, warmer colors are increasing height and dark blue colors are negative. A) The 2D spectrum for CFP-YFP FRET can be decomposed into the superposition of CFP (ΦC,C), YFP (ΦY,Y) and CFP-YFP FRET (ΦC,Y). Recovering ε and s for each fluorophore in the system allows calculation of the unmixing matrix, A which can be linearly unmixed to estimate the fluorescence from CFP (xCFP), YFP (xYFP) and the FRET sensitized emission (xCY). B can be obtained by calibration with known FRET efficiency standards. Linear unmixing with B to allows estimation of concentrations of total fluorophores ([CFP] and [YFP]) and apparent FRET (ECY[CFP-YFP]) which are contained in vector c. During this step, a negative component (blue color) couples the FRET-associated decrease in donor fluorescence to an increase in acceptor fluorescence. B) For most instruments, the complete landscape is not measured, rather, excitation and emission bandpass filters (boxes) define portions of the excitation-emission landscape. For 2-Way FRET the three images needed are dc,c, dy,y and dc,y. C) As more fluorophores are added to the system (e.g. the addition of RFP), the spectral landscape grows by the addition of direct fluorescence components along the diagonal (d1,1, d2,2, and d3,3) and their possible FRET interactions which appear as off-diagonal peaks (e.g. d1,2, d1,3 and d2,3). D) The mathematical form of this problem generalizes to account for multiple fluorophores engaged in FRET.
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pone-0064760-g001: N-Way FRET on the excitation-emission landscape.Spectroscopically, FRET is a coupling between donor excitation and acceptor emission. This excitation-emission coupling (Φ) can be described by the outer product of the excitation vector ε and an emission vector s. The Φ signatures define the spectral library A for the N-Way FRET linear unmixing problem (d = Ax = Bc) that can be viewed on the 2D excitation-emission landscape in addition to viewing the data (d). Specifically, these appear as topographical features with light green = 0, warmer colors are increasing height and dark blue colors are negative. A) The 2D spectrum for CFP-YFP FRET can be decomposed into the superposition of CFP (ΦC,C), YFP (ΦY,Y) and CFP-YFP FRET (ΦC,Y). Recovering ε and s for each fluorophore in the system allows calculation of the unmixing matrix, A which can be linearly unmixed to estimate the fluorescence from CFP (xCFP), YFP (xYFP) and the FRET sensitized emission (xCY). B can be obtained by calibration with known FRET efficiency standards. Linear unmixing with B to allows estimation of concentrations of total fluorophores ([CFP] and [YFP]) and apparent FRET (ECY[CFP-YFP]) which are contained in vector c. During this step, a negative component (blue color) couples the FRET-associated decrease in donor fluorescence to an increase in acceptor fluorescence. B) For most instruments, the complete landscape is not measured, rather, excitation and emission bandpass filters (boxes) define portions of the excitation-emission landscape. For 2-Way FRET the three images needed are dc,c, dy,y and dc,y. C) As more fluorophores are added to the system (e.g. the addition of RFP), the spectral landscape grows by the addition of direct fluorescence components along the diagonal (d1,1, d2,2, and d3,3) and their possible FRET interactions which appear as off-diagonal peaks (e.g. d1,2, d1,3 and d2,3). D) The mathematical form of this problem generalizes to account for multiple fluorophores engaged in FRET.

Mentions: In this linear unmixing method for FRET microscopy, we use Parallel Factor analysis (PARAFAC) of single fluorophore samples to determine the instrument specific excitation and emission signatures for individual fluorophores. These signatures are then used to compose the possible excitation and emission couplings (EEC) both within (e.g. excitation and emission for one fluorophore) and between fluorophores (e.g their FRET-couplings, excitation of one fluorophore and emission of another, Fig. 1). These EECs are then used to generate a spectral library that when inverted, provides the least-squares estimate of the fluorescence contributions from each fluorophore and each FRET interaction in the system. A second calibration step uses linked FRET constructs to unitize the EECs and couples FRET-associated losses of donor fluorescence to increases in acceptor fluorescence. The result is a linear unmixing model that allows estimation of total fluorophore concentrations and apparent FRET efficiencies (product of the fraction of energy transferred and the fraction of interacting molecules). Extending this linear formalism, we provide a simple method for estimation of uncertainty based on propagation of shot noise into the fluorescence estimates, concentration estimates and apparent FRET efficiency estimates.


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 on the excitation-emission landscape.Spectroscopically, FRET is a coupling between donor excitation and acceptor emission. This excitation-emission coupling (Φ) can be described by the outer product of the excitation vector ε and an emission vector s. The Φ signatures define the spectral library A for the N-Way FRET linear unmixing problem (d = Ax = Bc) that can be viewed on the 2D excitation-emission landscape in addition to viewing the data (d). Specifically, these appear as topographical features with light green = 0, warmer colors are increasing height and dark blue colors are negative. A) The 2D spectrum for CFP-YFP FRET can be decomposed into the superposition of CFP (ΦC,C), YFP (ΦY,Y) and CFP-YFP FRET (ΦC,Y). Recovering ε and s for each fluorophore in the system allows calculation of the unmixing matrix, A which can be linearly unmixed to estimate the fluorescence from CFP (xCFP), YFP (xYFP) and the FRET sensitized emission (xCY). B can be obtained by calibration with known FRET efficiency standards. Linear unmixing with B to allows estimation of concentrations of total fluorophores ([CFP] and [YFP]) and apparent FRET (ECY[CFP-YFP]) which are contained in vector c. During this step, a negative component (blue color) couples the FRET-associated decrease in donor fluorescence to an increase in acceptor fluorescence. B) For most instruments, the complete landscape is not measured, rather, excitation and emission bandpass filters (boxes) define portions of the excitation-emission landscape. For 2-Way FRET the three images needed are dc,c, dy,y and dc,y. C) As more fluorophores are added to the system (e.g. the addition of RFP), the spectral landscape grows by the addition of direct fluorescence components along the diagonal (d1,1, d2,2, and d3,3) and their possible FRET interactions which appear as off-diagonal peaks (e.g. d1,2, d1,3 and d2,3). D) The mathematical form of this problem generalizes to account for multiple fluorophores engaged in FRET.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064760-g001: N-Way FRET on the excitation-emission landscape.Spectroscopically, FRET is a coupling between donor excitation and acceptor emission. This excitation-emission coupling (Φ) can be described by the outer product of the excitation vector ε and an emission vector s. The Φ signatures define the spectral library A for the N-Way FRET linear unmixing problem (d = Ax = Bc) that can be viewed on the 2D excitation-emission landscape in addition to viewing the data (d). Specifically, these appear as topographical features with light green = 0, warmer colors are increasing height and dark blue colors are negative. A) The 2D spectrum for CFP-YFP FRET can be decomposed into the superposition of CFP (ΦC,C), YFP (ΦY,Y) and CFP-YFP FRET (ΦC,Y). Recovering ε and s for each fluorophore in the system allows calculation of the unmixing matrix, A which can be linearly unmixed to estimate the fluorescence from CFP (xCFP), YFP (xYFP) and the FRET sensitized emission (xCY). B can be obtained by calibration with known FRET efficiency standards. Linear unmixing with B to allows estimation of concentrations of total fluorophores ([CFP] and [YFP]) and apparent FRET (ECY[CFP-YFP]) which are contained in vector c. During this step, a negative component (blue color) couples the FRET-associated decrease in donor fluorescence to an increase in acceptor fluorescence. B) For most instruments, the complete landscape is not measured, rather, excitation and emission bandpass filters (boxes) define portions of the excitation-emission landscape. For 2-Way FRET the three images needed are dc,c, dy,y and dc,y. C) As more fluorophores are added to the system (e.g. the addition of RFP), the spectral landscape grows by the addition of direct fluorescence components along the diagonal (d1,1, d2,2, and d3,3) and their possible FRET interactions which appear as off-diagonal peaks (e.g. d1,2, d1,3 and d2,3). D) The mathematical form of this problem generalizes to account for multiple fluorophores engaged in FRET.
Mentions: In this linear unmixing method for FRET microscopy, we use Parallel Factor analysis (PARAFAC) of single fluorophore samples to determine the instrument specific excitation and emission signatures for individual fluorophores. These signatures are then used to compose the possible excitation and emission couplings (EEC) both within (e.g. excitation and emission for one fluorophore) and between fluorophores (e.g their FRET-couplings, excitation of one fluorophore and emission of another, Fig. 1). These EECs are then used to generate a spectral library that when inverted, provides the least-squares estimate of the fluorescence contributions from each fluorophore and each FRET interaction in the system. A second calibration step uses linked FRET constructs to unitize the EECs and couples FRET-associated losses of donor fluorescence to increases in acceptor fluorescence. The result is a linear unmixing model that allows estimation of total fluorophore concentrations and apparent FRET efficiencies (product of the fraction of energy transferred and the fraction of interacting molecules). Extending this linear formalism, we provide a simple method for estimation of uncertainty based on propagation of shot noise into the fluorescence estimates, concentration estimates and apparent FRET efficiency estimates.

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