Presynaptic actin cytoskeleton at the Drosophila larval NMJ. (A) Diagram of the Drosophila larval neuromuscular system as a tissue mechanics model. The NMJ at muscle 4 (gray) was predominantly assessed in this study. The presynaptic compartment (light brown) is subjected to multiple mechanical forces including axonal tension (brown arrow) and compression due to muscle contractions (blue arrows). (B) Single frame from live imaging of presynaptic actin assemblies visualized via the neuronal expression of the genetically encoded actin marker mNeonGreen-GMA (mNGMA) in larval preparations with intact brains (see also Video 1). WEKA segmentation was used to distinguish different actin subpopulations (round (spot-like) and linear (cable-like or core) F-actin structures) and to facilitate their subsequent quantitative analysis (see also Fig. S1). Orange arrows indicate a linear actin core traversing the NMJ. (C) Single frame from live imaging of NMJs expressing an independent genetically encoded actin marker Lifeact::Halo delineates similar spot-like and cable-like (core) actin assemblies. Orange arrows indicate the linear actin core. (D) Single frame from live imaging of the Drosophila actin isoform 5C, tagged with the red fluorescent protein mScarlet-I (QAct5C::mScarI), similarly delineates the actin core in addition to round F-actin assemblies. (E) Image sequence of FRAP of QAct5C::mScarI-labeled actin core. (F) FRAP curve of QAct5C::mScarI (N—NMJs; error bars represent the SEM). Scale bars—5 µm in A, 2 µm in other images. See Table S1 for detailed genotypes.