Association of the F-actin–stabilizing protein tropomyosin with linear F-actin structures in the presynaptic core. (A and B) Inverted contrast single frame from live imaging depicting the localization pattern upon neuronal expression of mNeonGreen-tagged Tm1-L (mNG-Tm1-L or “long” Tm1) and Tm1-A (mN-Tm1-A or “short” Tm1) in B. (C) F-actin stabilizer Tm1-A (mNG-Tm1-A) is enriched in the linear Lifeact::Halo-marked actin assemblies along the presynaptic core. (D) Pearson’s R correlation between Lifeact::Halo and GFP or mNG-Tm1-A. (E and F) Presynaptic localization of mNGMA-labeled actin assemblies and (F) mNG-Tm1-L is sensitive to treatment with LatA, and not the vehicle DMSO, supporting F-actin-Tm1 association with the presynaptic actin core. (G) Endogenous Tm1 (cyan), detected via immunofluorescence, traverses the NMJ (labeled with HRP—magenta) in larvae expressing control RNAi in all neurons. The Tm1 signal is significantly decreased at NMJs of animals neuronally expressing a Tm1 RNAi line targeting all known Tm1 isoforms (panTm1⁴³⁵⁴²), and the complementary line Tm1⁵⁶⁸⁶⁹, targeting the Tm1 isoforms A, Q, and R. (H) Quantification of the Tm1-A/L fluorescence per µm² at NMJs from larvae neuronally expressing CTRL, Tm1⁴³⁵⁴², or Tm1⁵⁶⁸⁶⁹ RNAi lines. Ns—nonsignificant, ***P < 0.001 and ***P < 0.001 upon the unpaired, nonparametric Mann–Whitney test, or after one-way ANOVA with the Kruskal–Wallis multiple comparisons test. N in bar graphs—NMJs. Error bars are the SEM. Scale bars—2 µm. See Table S1 for detailed genotypes and N.