Presynaptic actin cytoskeleton at the larval Drosophila NMJ. (A) Time-lapse insets (from Video 1) showing the structure and lifetime of linear F-actin/cable-like structures at the NMJ. (B) Time-lapse insets of round presynaptic F-actin assemblies (encircled) with different lifetimes (green circle around a longer lived assembly, light-blue circle around a short-lived, possibly endocytic event) visualized via the actin marker QmNGMA at the NMJ. (C) Time-lapse of FRAP of the neuronally expressed actin marker QmNGMA at a bouton containing linear F-actin assemblies. The bleached bouton is outlined in yellow. (D) FRAP quantification of linear F-actin–containing region showing recovery of QmNGMA (1 frame/s, n = 3). (E) Inverted contrast montage of the maximum intensity projection, along with individual Z-stacks of an NMJ from a larva expressing mScarlet-I::Act5C neuronally (nSybQF>QAct5C::mScarI). The yellow rectangle marks a typical region of the NMJ selected for FRAP analysis of the presynaptic actin core. (F) Presynaptic actin assemblies are sensitive to a treatment with the actin depolymerization–inducing drug LatA. (G) Quantifications of the fluorescence per µm² and CoV for the QmNGMA actin marker in DMSO and LatA-treated larval fillets, measured through the middle of NMJs, where the actin core predominantly traverses. Error bars represent the SEM. N in bar graphs—NMJs. ns—nonsignificant, *P < 0.05, **P < 0.01, and ***P < 0.001 upon the unpaired, nonparametric Mann–Whitney test. Scale bars—2 µm. See Table S1 for detailed genotypes and N. CoV, coefficient of variation.