Figure 3.

HaloTag-based SV labeling enabled quantifying the fraction of recycled SVs. A, Diagram represents HaloTag labeling with HL-Cy5 during recycling of SVs carrying the VpHalo. B, Diagram represents the timeline of the labeling experiment. Fish preparations were subjected to incubation with HL-Cy5 (magenta square) combined with HK depolarization in the absence (boxed in black; –Ca²⁺) or presence (boxed in orange; +Ca²⁺) of [Ca²⁺]_o. In parallel, fish after fixation were incubated overnight in PBST containing HL-Cy5 (boxed in gray; full labeling). C, Confocal images of TagRFP, pHluorin (pH 7.4), and Cy5 at the NMJs of fish stimulated by HK for 5 min in the absence (–Ca²⁺) or presence (+Ca²⁺) of [Ca²⁺]_o. Scale bar, 10 μm. D, E, Relationship between pHluorin fluorescence (F_{pH (pH7.4)}) and Cy5 fluorescence (F_cy5) measured at individual ROI in full labeling (D) and HK-stimulated (E) conditions. F, Average F_cy5/F_{pH (pH7.4)} normalized to that of the value in a full labeling condition (n = 10 fish). +Ca²⁺ stimulation for 5 min (n = 10 fish), 10 min (n = 10 fish), and 15 min (n = 10 fish) mobilized larger SV fractions than –Ca²⁺ stimulation (n = 6 fish). *Adjusted p < 0.05, ***adjusted p < 0.001, one-way ANOVA followed by Bonferroni–Holm test. Error bars indicate ±SEM.