Journal: bioRxiv
Article Title: Topology-dependent FRET efficiency in living cells via N-C swapping of fluorescent protein fusions
doi: 10.64898/2026.06.03.729767
Figure Lengend Snippet: a, b. Raw ( top ) and pseudocolored ( bottom ) confocal images of donor fluorescent protein (a: EGFP or b: EYFP) in COS-1 cells expressing the indicated fluorophore combinations before ( Pre ) and after ( Post ) photobleaching of acceptor protein (mCherry). COS-1 cells were transiently transfected with EGFP-mCherry and mCherry-EGFP (a) or EYFP-mCherry and mCherry-EYFP (b) expression vectors. As negative controls, the EGFP (a) or EYFP (b) expression vector was co-transfected with an expression vector encoding non-fused mCherry. After transfection, live-cell imaging was performed using a confocal laser scanning microscope. Experiments were repeated at least three times. Pseudocolor bars indicate fluorescence intensity, with H and L representing high and low signal intensity, respectively. Bar = 20 µm. c. FRET efficiency determined by acceptor photobleaching in COS-1 cells expressing EGFP-mCherry (n = 31), mCherry-EGFP (n = 34), EYFP-mCherry (n = 28), or mCherry-EYFP (n = 33). As negative controls, COS-1 cells expressing non-fused combinations of EGFP and mCherry (n = 28) or EYFP and mCherry (n = 27) were analyzed. Data were obtained from three independent experiments. d, e. FRET efficiencies determined by live-cell acceptor photobleaching in the nucleus or cytoplasm of COS-1 cells expressing EGFP-mCherry (n = 31 each for nucleus and cytoplasm) and mCherry-EGFP (n = 34 each for nucleus and cytoplasm each) (D), or EYFP-mCherry (n = 28 each for nucleus and cytoplasm) and mCherry-EYFP (n = 33 each for nucleus and cytoplasm) (E). Data were obtained from three independent experiments. DA , donor-acceptor configuration (EGFP-mCherry and EYFP-mCherry); AD , acceptor-donor configuration (mCherry-EGFP and mCherry-EYFP); D/A , non-fused control of donor (EGFP or EYFP) and acceptor (mCherry); Nuc , nucleus; Cyt , cytoplasm. Values are expressed as means ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two-way ANOVA followed by Bonferroni/Dunn’s post- hoc tests. n.s., not significant.
Article Snippet: COS-1 cells were originally obtained from ATCC (Manassas, VA, USA) and kindly provided by the laboratory at Department of Anatomy and Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine (Kyoto, Japan).
Techniques: Expressing, Transfection, Plasmid Preparation, Live Cell Imaging, Laser-Scanning Microscopy, Fluorescence, Control