Fig. 1: Spatiotemporal visualization of tryptophan in vitro and in vivo with ratiometric GRIT sensor.

a GRIT sensor allows quantitative measurements of tryptophan in cultured cells and zebrafish. Design and possible working mechanism of GRIT sensor (top). GRIT sensor is composed of TrpR (PDB ID: 1JHG, shown in cyan) and cpSFYFP (PDB ID: 3EVP, shown in green). Tryptophan (Trp, shown in magenta) binding to GRIT sensor elicits a large fluorescence change, enabling quantitative measurements of tryptophan (bottom). b Excitation spectra of purified GRIT in the absence or presence of 1 mM Trp, normalized to the major peak intensity at 1 mM Trp. Emission was measured at 530 nm. Two excitation peaks were at 420 nm (Ex1) and 500 nm (Ex2), respectively (indicated by arrows). c Excitation ratios of GRIT and GRITOL in the presence of different concentrations of Trp. d Fluorescence images of HeLa cells expressing cytosolic GRIT (left) or GRITOL (right) in respond to Trp or His in HBSS buffer. Images are pseudocolored with the ratio of excitation fluorescence at 488 nm and 405 nm (R_488/405). Scale bars, 10 μm. e Time course of averaged fluorescence intensity of GRIT in cytosol in response to exogenous Trp or His of absolute concentrations in HBSS buffer. f The maximum dynamic ranges were plotted against Trp or His concentrations. g Kinetics of GRIT responses in HeLa cells upon addition of 1 mM indicated amino acids. h Bidirectional responses of GRIT sensor to the addition of 5 μM Trp and a followed addition of 1 mM indicated amino acids. i Representative images of GRIT sensor driven by beta-actin in response to 5 mM Trp or 20 mM His. Scale bars, 50 μm. j, k Group analysis of GRIT responses to Trp (j) or His (k) (n = 4). l, m Representative images (l, maximum projection along the z-axis) and averaged kinetics (m) of GRIT signals in brain, spinal cord and muscles of zebrafish treated with LPS or PBS (n = 6–8). Scale bars, 50 μm. n, o Representative fluorescence images (n) and mean ratios (o) of GRIT protein in plasma of zebrafish caudal vein (CV) and dorsal aorta (DA) with the indicated treatments (n = 3–5). Scale bars, 50 μm. p The quantification measurements of tryptophan levels and biophysical models of tryptophan in mammalian cells (top) and zebrafish (bottom). Tryptophan enters cells by SLC7A5 (LAT1) and exits from cells in the present of Class I and II amino acids, which could be inhibited by JPH203 (top). During LPS-induced inflammation, plasma tryptophan enters the brain, spinal cord, and muscles (bottom). SLC, solute carrier. Data shown as means ± sem. (n.s. not significant; ****P < 0.0001). n = 3–6 independent experiments (b–h). Two-way ANOVA (m) and Student’s unpaired t-test for (j, k, o).