Background: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser.
Methods: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564-606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters. Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached. Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry.
Results: Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd. Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564-606 nm. A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564-606 nm, consistent with the conclusion that FRET was detected comparably by both methods. A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR --> YFP-FBPcd. FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached. The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry.
Conclusion: These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser. The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique.
Published 2003 Wiley-Liss, Inc.