Abstract
Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data.
Highlights
The protein-protein interactions mediate the propagation of information through biochemical signaling pathways
During the development of N-Way Fluorescence resonance energy transfer (FRET), we demonstrated that the spectral contributions of each species in the data, g, could be qualitatively unmixed into arbitrary units and either FRET or no FRET, x, as, x 1⁄4 AÀ1g ð3Þ
To validate the 3D-3Way FRET algorithm, reconstructions were performed on simulated data with defined fluorescence distributions
Summary
The protein-protein interactions mediate the propagation of information through biochemical signaling pathways. Fluorescence resonance energy transfer (FRET) microscopy methods are well suited for imaging the subcellular distributions of these protein-protein interactions in live cells[1]. FRET occurs when a donor’s emission spectrum and an acceptor’s excitation spectrum overlap; a requirement that comes at the cost of spectral mixing of fluorescent proteins with short Stokes shifts[2,3]. We previously developed N-Way FRET[4] to linearly unmix overlapping FRET signatures and recover the concentrations and apparent FRET efficiencies of two or PLOS ONE | DOI:10.1371/journal.pone.0152401. Findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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