Stretching of larval axons rearranges integrin receptors at the NMJ. (A) Diagram of Drosophila fillet preparation upon gentle pulling of the larval brain for 15 min to induce stretching of the axons and NMJs. This procedure should increase axonal tension exerted at presynaptic compartments. (B) mNGMA-labeled presynaptic actin at NMJs from fixed larvae with unstretched (left) and stretched brains (right). (C) Quantification of the fluorescence per µm² for the QmNGMA actin marker in whole NMJs. (D) Quantifications of the number of round F-actin assemblies per µm², as well as the area of the individual round presynaptic actin structures. (E) Graphs representing the percentage of NMJ area covered with linear F-actin, as well as the area of the individual assemblies. (F and G) Immunostaining of integrin-β (cyan) at NMJs of control larvae (C155-Gal4>CTRL³⁵⁷⁸⁵) after 15 min with unstretched and stretched brains and axons. (H) Integrin-β fluorescence in the HRP-delineated, postsynaptic, and muscle ROIs. (I) Quantifications of the abundance, size, and fluorescence of WEKA-segmented integrin-β structures along linear areas. Box-and-whisker graphs were used to represent the results of the area of the individual presynaptic actin structures, as well as the area and fluorescence intensity of integrin-β entities. 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 or integrin-β entities. ns—not significant, and ***P < 0.001 upon the unpaired, nonparametric Mann–Whitney test. Error bars represent the SEM. g—Hedges’ g represents the effect size. Scale bar—2 µm. See Table S1 for detailed genotypes and N.