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. 2010 Feb 22;5(2):e9344.
doi: 10.1371/journal.pone.0009344.

A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells

Carina Banning  1 , Jörg Votteler, Dirk Hoffmann, Herwig Koppensteiner, Martin Warmer, Rudolph Reimer, Frank Kirchhoff, Ulrich Schubert, Joachim Hauber, Michael Schindler
Affiliations

A flow cytometry-based FRET assay to identify and analyse protein-protein interactions in living cells

Carina Banning et al. PLoS One. .

Abstract

Background: Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.

Results: Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.

Conclusion: The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Setup of FRET-measurements by flow cytometry and microscopy.
(a) The experimental setup and gating strategy to measure FRET by FACS. Living 293T cells transfected with the controls CFPonly, YFPonly, CFP and YFP as well as the CFP-YFP fusion proteins were analysed on a FACS Aria flow cytometer. Double positive cells were gated (panel 1) and false positive FRET signals resulting from YFP excitation by the 405 nm laser were excluded (panel 2). The remaining cells were evaluated for FRET by adjusting a gate defining to cells which are cotransfected with CFP and YFP only and should thus be FRET-negative (panel 3). (b) Living 293T cells and cells from the same transfections were treated with 2% PFA and analysed for FRET as depicted in (a). Shown are mean values +/− standard deviation from seven independent transfections. (c) 293T cells were grown on cover slips and cotransfected with CFP and YFP or the CFP-YFP fusion protein and mounted on microscope slides. Confocal images were taken and analysed for FRET using the “FRET and colocalization analyzer” ImageJ plug-in (7). “FRET”-images give the calculated amount of FRET for each pixel in the merged images. The ImageJ plug-in colour codes the relative FRET efficiency which is indicated by the displayed colour bar. Furthermore the “coloc/FRET”-plots display pixel colocalization as well as colour coded FRET efficiency in a 2D plot. CFP is shown in red and YFP in green.
Figure 2
Figure 2. Analyses of protein interactions by FRET.
(a) Representative primary FACS-plots showing the amount of FRET+ cells in living 293T cells cotransfected with the indicated CFP and YFP fusion proteins. Numbers give total percentages of cells within the FRET gate (compare Fig. 1a panel 3). (b) Mean values and standard deviations (SD) for the total amount of FRET+ cells from a minimum of eight independent experiments that were analysed as depicted in (a). The dotted line gives the maximum background FRET-signal that was obtained when cells were cotransfected with the MEM-CFP control and the YFP fusion proteins. Abbreviations: CfY, CFP fused YFP; CaY, CFPonly and YFP-fusion. (c) Merged images from confocal pictures of 293T cells that were cotransfected with the Nef/Vpu-YFP fusion proteins (shown in green) and the indicated CFP-fusions (shown in red). Regions in which both fusions colocalize appear yellow.
Figure 3
Figure 3. Vpu interacts with CD317 via its transmembrane region.
(a) Representative primary FACS-plots showing the amount of FRET+ cells in living 293T cells cotransfected with the indicated CD4 or CD317-CFP and Vpu-YFP fusion proteins. (b) Mean values and standard deviations (SD) for the total amount of FRET+ cells from six independent experiments that were analysed as depicted in (a). (c) Confocal images of 293T cells that were cotransfected with the Vpu-YFP fusion proteins (shown in green) and either CD317-CFP or CD4-CFP (shown in red). (d) 293T cells were transfected with the indicated Vpu-YFP fusions and a FLAG-tagged CD317. Vpu immune complexes were isolated from cell detergent extracts by immunoprecipitation with anti-Vpu rabbit serum and analysed for the presence of CD317 by Western blot with anti-FLAG.
Figure 4
Figure 4. Measurement of HIV-1 Rev multimerization by FACS-FRET.
(a) Representative primary FACS-plots showing the amount of FRET+ cells in living 293T cells cotransfected with the indicated CFP and YFP fusion proteins. (b) Mean values and standard deviations (SD) for the total amount of FRET+ cells from four independent experiments that were analysed as depicted in (a). (c) Confocal images of 293T cells that were cotransfected with the indicated YFP (shown in green) and CFP (shown in red) fusion proteins.
Figure 5
Figure 5. High-throughput-screening for unknown protein interactions by FACS-FRET.
(a) Experimental setup to screen for unknown protein interaction with flow cytometry based FRET in high-throughput. (b) Living 293T cells were transfected with the Vpu-YFP fusion as a bait and a mixture of equal amounts of the CFP-fusion constructs that are described in Figure 2. 36 h post transfection FRET+ cells were sorted, pelleted, resuspended in PBS and reanalysed for successful purification. Abbreviation GOI, gene of interest.
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