Quantitative spectral Linear Unmixing and Ratiometric FRET for live-cell imaging of protein interactions

We present a biophysical imaging strategy based on linear unmixing Forster 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 HIGHLIGHTWe 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/673709v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@42beecorg.highwire.dtl.DTLVardef@4a746org.highwire.dtl.DTLVardef@181f61corg.highwire.dtl.DTLVardef@144d940_HPS_FORMAT_FIGEXP M_FIG C_FIG