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

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. Confocal live-cell images of donor fluorescent protein (a: EGFP or b: EYFP) and FRET signal channels in COS-1 cells expressing the indicated fluorophore combinations. 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 sensitized emission in living COS-1 cells expressing EGFP-mCherry (n = 31), mCherry-EGFP (n = 34), EYFP-mCherry (n = 27), or mCherry-EYFP (n = 33). COS-1 cells expressing non-fused combinations of EGFP and mCherry (n = 25) or EYFP and mCherry (n = 22) served as negative controls. Data were obtained from three independent experiments. d, e. FRET efficiencies determined by live-cell sensitized emission in the nucleus ( Nuc ) or cytoplasm ( Cyt ) of COS-1 cells expressing EGFP-mCherry (n = 31 each for nucleus and cytoplasm) and mCherry-EGFP (n = 34 each for nucleus and cytoplasm) (d), or EYFP-mCherry (n = 27 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 < 0.01, *** 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

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–d. Jittered plots of the transformed ( t ) FRET metric, K = E /(1- E ), calculated from FRET efficiency measured by live-cell acceptor photobleaching in COS-1 cells expressing the indicated fusion proteins: EGFP-mCherry (n = 30 each for nucleus and cytoplasm), mCherry-EGFP (n = 33 each for nucleus and cytoplasm), EYFP-mCherry (n = 28 each for nucleus and cytoplasm), and mCherry-EYFP (n = 33 each for nucleus and cytoplasm). Values are derived from three independent experiments. Data are shown as individual points (jittered). The dotted lines indicate the mean. **** p < 0.0001 by unpaired t -test. e. Orientation index ( OI = K AD / K DA ) in EGFP-mCherry and mCherry-EGFP, and EYFP-mCherry and mCherry-EYFP, calculated separately in the nucleus ( Nuc ) and cytoplasm ( Cyt ). Statistics were performed on mean values from three independent experiments (n = 3). Data are shown as means ± SE. ** p < 0.01 by one-sample t -test (µ = 1). The dotted line indicates 1.0. f–i. Scatter plots of donor fluorescence intensity ( FI ) vs. FRET efficiency measured by acceptor photobleaching in COS-1 cells expressing EGFP-mCherry (F; n = 173 ROIs), mCherry-EGFP (G; n = 181 ROIs), EYFP-mCherry (H; n = 151 ROIs), and mCherry-EYFP (I; n = 171 ROIs). Red dotted lines indicate linear regression fits ( r ² values shown). Data are from three independent experiments. Negligible correlations were observed.

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: Transformation Assay, Expressing, Derivative Assay, Fluorescence