Extended Data Fig. 1: Development of nLightR2 and nLightG2.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a, Partial alignment of the amino acid sequence of RdLight1 with rGRABDA3m. Regions grafted from RdLight1 in the engineering of nLightR are shown in pink. Red letters mark the five point mutations in rGRABDA3m relative to RdLight1. b–c, Dynamic range (mean±s.e.m.) of nLightR and combinatorial variants in response to NE (10 µM). The presence (green) or absence (white) of each mutation is indicated. d, Dynamic range (mean ± s.e.m.) of nLightR, nLightR1.1 (Q237K, H242M, A484H), and nLightR2 (Q237K, H242M, A484H, C-terminal truncation at Q595) in HEK293T cells following NE (10 µM). Two-tailed Student’s t-test with Welch’s correction: ****P = 7.878×10−5 (n = 42 cells, six experiments). e, Basal brightness and surface expression (mean±s.e.m.) of nLightR and nLightR2 in HEK293T cells by confocal microscopy. Expression was quantified by FLAG staining, and fluorescence intensities were normalized to nLightR (n = 6 ROIs, six experiments). f, Apparent molecular basal brightness (normalized by expression level) of nLightR and nLightR2; no significant difference (p = 0.402). g, Dynamic range (mean±s.e.m.) of nLightR2 and the inactive mutant nLightR2-ctr (D129A) in response to NE (10 µM); p = 0.239. h, Confocal image of HEK293T cells expressing nLightR2-ctr. Scale bar, 20 μm. Representative of four independent experiments. i, Partial alignment of dLight1.3b with gGRABDA3m. Grafted regions from dLight1.3b used for nLightG are in green. Red letters indicate six point mutations in gGRABDA3m. j, Dynamic range (mean±s.e.m.) of nLightG, nLightG1.1 (S239N, N243Y, K260G, D441W, N444F), and nLightG2 (same mutations plus C-terminal truncation at Q594) upon NE (10 µM). p = 2.891×10−3 (n = 21 cells, three experiments). k, Basal brightness and surface expression of nLightG, nLightG2, and GRABNE2m. l, Apparent molecular basal brightness of nLightG, nLightG2, and GRABNE2m; nLightG2 vs GRABNE2m: p = 5.918×10−4; nLightG vs nLightG2: p = 0.220. m, Dynamic range of nLightG2 and nLightG2-ctr (D129A); p = 0.256. n, Confocal image of HEK293T cells expressing nLightG2-ctr. Scale bar, 20 μm. Representative of four independent experiments.
