Quantitative spectral Linear Unmixing and Ratiometric FRET for live-cell imaging of protein interactions
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Abstract
ABSTRACT We present a biophysical imaging strategy based on linear unmixing Förster resonance energy transfer (lux-FRET) for investigating protein-protein interactions and receptor-mediated signaling in live cells. This method utilizes spectral unmixing of FRET signals acquired via confocal laser scanning microscopy (LSM), enabling high-resolution quantification of molecular interactions with both spatial and temporal precision. Applying lux-FRET, we examined receptor-receptor interactions and downstream signaling events, including agonist specificity for 5-HT receptors. Ratiometric FRET measurements with a genetically encoded cAMP biosensor allowed us to assess biosensor sensitivity to cyclic nucleotides and receptor efficacy. Additionally, we explored physiological interactions between CD44 and 5-HT receptors and characterized the oligomerization state of the 5-HT1A receptor through apparent FRET efficiency analysis. Our findings demonstrate the utility of lux-FRET combined with quantitative molecular microscopy as a powerful tool for dissecting dynamic signaling mechanisms in live cells. This approach offers broad applicability for researchers studying receptor pharmacology, cellular signaling, and protein interaction dynamics. RESEARCH HIGHLIGHT We present a real-time imaging strategy combining lux-FRET with quantitative molecular microscopy to study protein interactions and receptor signaling in living cells. Using spectral and ratiometric FRET analysis, this method enables high-resolution visualization of dynamic molecular processes under physiological conditions. GRAPHICAL ABSTRACT
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ABSTRACT
We present a biophysical imaging strategy based on linear unmixing Förster resonance energy transfer (lux-FRET) for investigating protein-protein interactions and receptor-mediated signaling in live cells. This method utilizes spectral unmixing of FRET signals acquired via confocal laser scanning microscopy (LSM), enabling high-resolution quantification of molecular interactions with both spatial and temporal precision. Applying lux-FRET, we examined receptor-receptor interactions and downstream signaling events, including agonist specificity for 5-HT receptors. Ratiometric FRET measurements with a genetically encoded cAMP biosensor allowed us to assess biosensor sensitivity to cyclic nucleotides and receptor efficacy. Additionally, we explored physiological interactions between CD44 and 5-HT receptors and characterized the oligomerization state of the 5-HT1A receptor through apparent FRET efficiency analysis. Our findings demonstrate the utility of lux-FRET combined with quantitative molecular microscopy as a powerful tool for dissecting dynamic signaling mechanisms in live cells. This approach offers broad applicability for researchers studying receptor pharmacology, cellular signaling, and protein interaction dynamics.
RESEARCH HIGHLIGHT We present a real-time imaging strategy combining lux-FRET with quantitative molecular microscopy to study protein interactions and receptor signaling in living cells. Using spectral and ratiometric FRET analysis, this method enables high-resolution visualization of dynamic molecular processes under physiological conditions.
Competing Interest Statement
The authors have declared no competing interest.
- ABBREVIATIONS
- CEpac
- Cerulean/Citrine tagged exchange protein activated by cAMP
- cNMP
- Cyclic nucleotide mono phopshate
- cAMP
- Cyclic adenosine mono phosphate
- cCMP
- Cyclic cytidine mono phosphate
- cGMP
- Cyclic gunanine mono phosphate
- cUMP
- Cyclic uridine mono phosphate
- cNMP-AM
- Cyclic nucleotide mono phosphate-acetoxymethyl ester
- cAMP-AM
- Cyclic adenosine mono phosphate-acetoxymethyl ester
- cCMP-AM
- Cyclic cytidine mono phosphate-acetoxymethyl ester
- cGMP-AM
- Cyclic guanine mono phosphate-acetoxymethyl ester
- cUMP-AM
- Cyclic uridine mono phosphate-acetoxymethyl ester
- FRET
- Förster Resonance Energy Transfer
- FSK
- Forskolin
- IBMX
- 3-isobutyl-1-methylxanthine
- Lux-FRET
- linear unmixing FRET
- LP-211
- N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide
- PKCα
- Protein kinase C alpha
- PMA
- 4 beta-phorbol 12-myristate 13-acetate
- PO4-AM3
- Phosphate tris (acetoxymethyl) ester
- WT
- Wild type
- 5-HT1AR
- Serotonin receptor subtype 1A
- 5-HT7R
- Serotonin receptor subtype 7
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- last seen: 2026-05-20T01:45:00.602351+00:00