Extended Data Fig. 3: Intracellular signaling characterization of nLightR2 and nLightG2.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a, Calcium signals in HEK293T cells expressing GCaMP6s with either wild-type sperm whale A1AAR (swA1AAR, left) or nLightR2 (right) upon sequential addition of NE (10 µM) and ionomycin (10 µM). GCaMP6s dynamic range was normalized to the ionomycin-induced maximal signal. Data from four independent experiments, n = 28 cells for both swA1AAR-GCaMP6s and nLightR2-GCaMP6s. b, Quantification of (a): normalized GCaMP6s response to NE (10 µM) with wt receptor vs. nLightR2. Two-tailed Student’s t-test with Welch’s correction, P = 1.808×10−22. c, Same as in (a), but for swA1AAR or nLightG2 co-expressed with the red calcium indicator jRGECO1a. Data from four experiments, n = 21 cells for swA1AAR-jRGECO1a and nLightG2-jRGECO1a. d, Quantification of (c): P = 3.071×10−23. e, Schematic of the NanoLuc complementation assay. Both receptor/indicator–SmBiT fusion and LgBiT–miniG fusion are exogenously expressed. Ligand-induced receptor activation recruits tagged miniG-proteins, restoring NanoLuc activity and generating luminescence. f, Time traces showing miniGq/s/i or β-arrestin2 recruitment to swA1AAR (gray), nLightG2 (green), or nLightR2 (magenta) after NE (10 µM) stimulation, or to hmGLP1R upon GLP1 (100 nM) stimulation. Recruitment causes NanoLuc complementation between SmBiT (fused to receptor) and LgBiT (fused to miniG or β-arrestin2). Luminescence was normalized to control (no ligand). Data are mean ± s.e.m.; n independent experiments: miniGq, nLightR2 (3), nLightG2 (3), swA1AAR (6); miniGs, nLightR2 (3), nLightG2 (3), swA1AAR (6); miniGi, nLightR2 (3), nLightG2 (3), swA1AAR (6); β-arrestin2, nLightR2 (6), nLightG2 (6), GLP1R (4). g, Statistical analysis of (f). Mean normalized luminescence (mean ± s.e.m.) after NE (10 µM) or GLP1 (100 nM) addition compared between swA1AAR/hmGLP1R and nLight sensors. Two-tailed Student’s t-test with Welch’s correction: miniGq, swA1AAR vs. nLightG2 ****p = 4.602×10−7, vs. nLightR2 ****p = 4.322×10−7; miniGs, ****p = 1.568×10−5 (nLightG2), ****p = 1.802×10−5 (nLightR2); miniGi, ****p = 2.307×10−5 (nLightG2), ***p = 6.508×10−4 (nLightR2); β-arrestin2, ****p = 1.418×10−10 (nLightG2), ****p = 6.026×10−13 (nLightR2).
