Presynaptic actin assemblies upon downregulation and overexpression of NMII ^HC /Zip in neurons. (A) Inverted contrast mNGMA-labeled presynaptic actin in fixed larvae co-expressing CTRL³⁵⁷⁸⁵ or NMII^HC/Zip⁶⁵⁹⁴⁷ RNAi lines in neurons. (B) Quantification of the fluorescence per µm² for the mNGMA actin marker in whole NMJs, along with the number of actin assemblies per µm², and the area and fluorescence intensity of the individual presynaptic actin structure. The actin particles were analyzed without specifying their morphological features as round or linear to account for fragmentation of the presynaptic cytoskeleton as a result of fixation¹. (C) Live-imaged inverted contrast Lifeact::Halo-labeled presynaptic actin in control larvae expressing GFP and larvae expressing NMII^HC/Zip::GFP. (D and E) Analyses of the F-actin fluorescence, as well as individual WEKA-segmented round and linear F-actin entities in GFP- and Zip::GFP-expressing animals. Box-and-whisker graphs were used to represent the results of the area and fluorescence intensity of the individual presynaptic actin structures. Whiskers represent 10^th to 90^th percentile, while the rest of the data points are shown as individual values. The y axis in these graphs represents log₁₀, to capture the broad distribution of the individual values. In bar graphs with linear y axis, error bars represent the SEM. N in bar graphs—NMJs; N in box and whiskers—individual actin assemblies. ns—nonsignificant, **P < 0.01, ***P < 0.001 upon the unpaired, nonparametric Mann–Whitney test. Scale bars—2 µm. See Table S1 for detailed genotypes and N.