Elastic modularity of AZ nanostructure revealed by STED imaging. (A) Representative STED images of AZs in wild-type, endo^RNAi, and rab3 mutant NMJs labeled with anti-BRP and anti-Unc13A. (B–D) BRP (B) and Unc13A (C) ring diameters are reduced in endo^RNAi and enhanced in rab3 mutants, while the BRP:Unc13A ratio remains the same (D). (E) Quantal modules of BRP clusters within single AZs are revealed by STED analysis using an averaging algorithm (Böhme et al., 2019). The three modules of peak frequency are shown for wild type (4, 5, and 6 modules), endo^RNAi (2, 3, and 4), and rab3 (8, 10, and 12). (F–H) Quantification of the mean number of BRP (F) and Unc13A (G) modules per AZ is shown, with a reduction in endo^RNAi and enhancement in rab3, while the ratio is largely unchanged (H). (I and J) Cumulative probability curve (I) and binned histogram (J) of BRP modules per NMJ are shown, with a leftward distribution observed in endo^RNAi and rightward shift in rab3 mutants. Error bars indicate ± SEM (n ≥ 53; one-way ANOVA, K-S test for I; Table S1). *, P < 0.05; ****, P < 0.0001; ns, not significant.