Fig. 5: Luminal pH alterations are important to crystal formation within the iridosome.
From: pH variations enable guanine crystal formation within iridosomes
a, Confocal microscopy images of several iridophores within zebrafish larvae eye at 96 hpf stained with LysoSensor (LysoS), which exhibits increased fluorescence in acidic environments. Top, maximum intensity projection image from a z stack showing iridophore intracellular acidic compartments (green) and crystals (white) within the iridophore. Right, zoomed-in view. Bottom, heat map representation of a single plane of the z stack. b, Box-and-whisker plot showing the fluorescent intensity of ROIs from small <1 μm2 iridosomes (n = 505, green) compared to medium 1–5 μm2 (n = 769, pink) and >5 µm2 iridosomes (n = 601, blue). The mean values ± s.d. are shown. The ROI mean values were compared using a two-sided linear mixed model, with classification group as a fixed factor and fish ID (n = 4) and crystal ID (n = 120) as random factors. P = 0.0231276 for 1–5 μm2 versus <1 μm2; P = 0.0000004 for <1 μm2 versus >5 μm2 and P = 0.0043115 for >5 μm2 versus 1–5 μm2. The horizontal lines mark the maximum, median and minimum values of the data, boxes mark the upper and lower quartiles and data points beyond this range are plotted as outliers. c, Graphs depicting the reflectance coverage area (top) and the Lysotracker fluorescence signal (bottom) in the eyes of larvae (blue) treated with bafilomycin A1 (n = 9) and lansoprazole (n = 10) compared to control larvae (purple) treated with DMSO (n = 10 and 11, respectively). The mean values ± s.d. are shown. Each data point represents an independent biological replicate. Statistical analysis was performed using a two-way ANOVA, with crystal reflectance and fluorescence coverage areas log-transformed before analysis. P = 1.83 × 10−15 for bafilomycin reflectance, P = 3.02 × 10−12 for lansoprazole reflectance, P = 4.2 × 10−14 for bafilomycin fluorescence and P = 6.27 × 10−5 for lansoprazole fluorescence compared to DMSO control. d,e, Confocal microscopy z-stack images of eye iridophores from 72 hpf zebrafish larvae treated with DMSO (control) or treated with 50 nM bafilomycin A1 (d) or 250 μM lansoprazole (e). Insets, higher-magnification views of areas within an iridophore. Lysotracker staining (LysoT, magenta) and crystal reflectance (Ref, white) are shown.
