Distribution of endogenous and neuronally expressed NMII ^HC /Zip at Drosophila larval NMJs. (A) Immunostaining of endogenous NMII^HC/Zip (cyan) at NMJs (HRP, magenta) of wild-type larvae labels large and small puncta at the NMJ and in muscles (yellow arrows—large Zip; white arrows—small Zip structures). Smooth, aggregate-like Zip puncta are visible predominantly in muscles (gray arrows). (B) Zip puncta (cyan) in distal axonal bundles (HRP in magenta) in animals neuronally (C155-Gal4) expressing CTRL³⁵⁷⁸⁵, NMII^HC/Zip⁶⁵⁹⁴⁷, and NMII^LC/Sqh³²⁴³⁹ RNAi lines. (C) Quantification of Zip fluorescence in axonal bundles. (D) Live imaging of neuronally co-expressed NMII^HC/Zip::GFP (cyan) and Lifeact::Halo (magenta) similarly reveals Zip puncta with different sizes at the NMJ, some of which are enriched at spot-like and linear F-actin assemblies (see also Video 6). (E) Independent examples of live-imaged NMJs of larvae expressing Zip::GFP and Lifeact::Halo. White arrows point to putative bipolar Zip filaments. (F) Pearson’s R correlation between Lifeact::Halo and Zip::GFP. (G) Quantification of the abundance and size of smaller and larger Zip puncta at the NMJ, as well as the average distance between adjacent Zip puncta in putative bipolar Zip filaments. In bar graphs with linear y axis, error bars represent the SEM. N in bar graphs—NMJs; N in box and whiskers—individual Zip assemblies. ***P < 0.001 upon the unpaired, nonparametric Mann–Whitney test. Scale bars—2 µm. See Table S1 for detailed genotypes and N.