Journal: Molecular Metabolism

Article Title: Development of a genetically encoded melanocortin sensor for high sensitivity in vivo imaging

doi: 10.1016/j.molmet.2025.102254

Figure Lengend Snippet: Generation and optimization of the FLARE MC sensors . ( A ) Schematic diagram of circular permutated enhanced green fluorescent proteins (cpGFP)-based GPCR sensors, including the FLARE MC . Image was created with www.biorender.com . ( B ) Cellular localization of MC1R and MC4R receptor tagged with EGFP in their C-terminus, at basal state. HEK293 cells were transfected with either MC1R-EGFP or MC4R-EGFP constructs, and confocal microscopy was conducted 48 h post-transfection. ( C ) Alignment of eCB2.0 sensor cpGFP junction with ICL3 junction of mMC1R (Arg 5.74 from hMC1R). Vertical lines indicate where cpGFP and linker were introduced. ( D ) Confocal image of MC1R containing GRAB eCB2.0 's cpGFP with flanking regions including ICL3 (left). Cells were transfected with MC1R with its ICL3 replaced with cpGFP with flanking regions of either eCB2.0, DA2m, or NE1m sensor. Shown are total fluorescence in cells at basal (control) and with 10 μM MTII treatment (right). ( E ) Saturation-mutagenesis with amino acid substitutions at position 18, 83, and 136 of cpGFP. F 0 values were normalized to those of the GRAB DA2m sensor. Shown are the screened mutants, including FLARE MC1.0 and FLARE MC1.1 , their responses at each position, and their overall ranking based on increased fluorescence. Scale bars ( B and D ), 10 μm.

Article Snippet: 0 -IRES-mCherry-CAAX [ ] (addgene #164611), and pAAV-hSyn-GRAB NE1m [ ] (addgene #123308) were obtained from addgene ( www.addgene.org , Watertown, MA, USA).

Techniques: Transfection, Construct, Confocal Microscopy, Fluorescence, Control, Mutagenesis