The activator protein-1 transcription factor is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper proteins. We have previously shown by fluorescence cross-correlation spectroscopy (FCCS) that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, cotransfected in HeLa cells, formed stable complexes in situ. Here we studied the relative position of the C-terminal domains via fluorescence resonance energy transfer (FRET) measured by flow cytometry and confocal microscopy. To get a more detailed insight into the conformation of the C-terminal domains of the complex we constructed C-terminal labeled full-length and truncated forms of Fos. We developed a novel iterative evaluation method to determine accurate FRET efficiencies regardless of relative protein expression levels, using a spectral- or intensity-based approach. The full-length C-terminal-labeled Jun and Fos proteins displayed a FRET-measured average distance of 8 +/- 1 nm. Deletion of the last 164 amino acids at the C-terminus of Fos resulted in a distance of 6.1 +/- 1 nm between the labels. FCCS shows that Jun-mRFP1 and the truncated Fos-EGFP also interact stably in the nucleus, although they bind to nuclear components with lower affinity. Thus, the C-terminal end of Fos may play a role in the stabilization of the interaction between activator protein-1 and DNA. Molecular dynamics simulations predict a dye-to-dye distance of 6.7 +/- 0.1 nm for the dimer between Jun-mRFP1 and the truncated Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full-length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. However, from our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm.

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. V C 2013 International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; FRET; laser scanning cytometry; high throughput; protein-protein interactions FLUORESCENCE or F€ orster resonance energy transfer (FRET) is a nonradiative process, in which energy is transferred from an excited fluorescent donor dye to a neighboring acceptor within its 2-10 nm vicinity by dipole-dipole coupling (1). In order for FRET to take place, the emission spectrum of the donor should overlap with the absorption spectrum of the acceptor, and the two dyes should have a proper relative orientation (2,3). The process is characterized by the FRET efficiency, E, which is the probability that a donor in the excited state transfers its energy to a nearby acceptor. E depends on the 6th power of the separation distance, R, between the donor and the acceptor:where R 0 is the F€ orster radius, at which E 5 0.5. Because of its sensitive distance dependence, FRET can be used as a molecular ruler to assess intra-or inter-molecular distances (4), molecular conformation or association state. FRET has several effects ...

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