Expression pattern and subcellular localization of SNT-3 in cholinergic motor neurons. (A and B) Domain structure and sequence alignment of the SNT-1 and SNT-3. The transmembrane domain (green), the C2A domain (light blue), and the C2B domain (light red) are highlighted. (C1–C6) Expression of snt-3 driven by the endogenous snt-3 promoter in various regions (arrow, neuron; star, muscle), including head neurons, tail neurons, motor neurons, head muscles, and body wall muscles. Scale bar, 50 µm. (D1–D6) Expression snt-3 (driven by the snt-3 promoter; red) in both cholinergic and GABAergic motor neurons (driven by the unc-17 and unc-25 promoters, respectively; green) at the ventral nerve cord. Scale bar, 20 µm. (E1–E6) Fluorescent images of the dorsal nerve cord (under the unc-129 promoter) showing colocalization of SNT-3::mApple with Rab3::GFP (SV marker) and SNT-1::GFP. Scale bar, 5 µm. (F1–F4) Expression patterns of SNT-1::mApple and SNT-3::mApple in WT and unc-13(s69) mutant background, respectively. Scale bar, 5 µm. (F5) Averaged percentage of the bouton fluorescence to the total axonal fluorescence of SNT-1::mApple and SNT-3::mApple in WT and unc-13 mutants. Data are mean ± SEM. ***P < 0.001. n.s., nonsignificant (one-way ANOVA). (G1–G4) Distribution of SNT-1::mApple and SNT-3::mApple in the dorsal or ventral nerve cords of WT and unc-104 KIF1A mutants are compared. SNT-1::mApple axonal fluorescence (dorsal nerve cord [DNC]) decreased, while cell body fluorescence (ventral nerve cord [VNC]) increased in unc-104 mutants, whereas SNT-3::mApple axonal fluorescence and cell body fluorescence were comparable between WT and unc-104 mutants. Stars indicate the cell bodies in VNC, and arrows indicate axons in DNC. Scale bar, 5 µm. (G5) Quantification of somatic and axonal fluorescence intensity of SNT-1::mApple and SNT-3::mApple in WT and unc-104 mutants. Data are mean ± SEM. ****P < 0.0001. n.s., nonsignificant (one-way ANOVA).