Fig. 6: Muscle contraction regulation of MN confinement is necessary for acute structural plasticity.

a Analysis of muscular inhibition by mechanical stretching. Schematic (left) of the protocol applied to larvae to achieve different degrees of stretching, where the same larvae is dissected to three successive conditions (1) HL3.1 relaxed (unstimulated), (2) high-K⁺ relaxed (stimulated relaxed) and (3) high-K⁺ stretched (stimulated stretched). Example of an image (right) of body-wall muscles visualized by expression of Tropomyosin-GFP, focusing on ventral muscles in segments A2-A4. The plotted line indicates the location used to create the kymographs shown below, which display the movement of the ventral muscles in each condition throughout time. Scale bar, 50 µm in body-wall muscles image and kymograph (over 300 sec). b Representative plot of muscle 6 length in segment A3 over time for each condition. Graphs were normalized by dividing the muscle length by maximal muscular extension. Top: unstimulated relaxed, middle: stimulated relaxed, bottom: stimulated stretched conditions. c, d Plots of mean and maximal contractility of muscle 6/segment A3. Lines connect the values obtained for individual larvae under each condition. n = 11 larvae. Statistical significance determined by one-way parametric analysis of variance (ANOVA); ****p < 0.0001, ns non-significant. e Images of control and MyoII-K/D animals in which muscle contraction was either allowed (relaxed fillets) or restricted (stretched fillets) after high-K⁺ stimulation. Presynaptic and postsynaptic membranes labeled with antibodies against Horseradish peroxidase (HRP-gray) and Discs-large (Dlg-green), respectively. Ghost boutons (GBs) (white arrow) are identified by the lack of Dlg. Scale bar, 10 µm. f Boxplot (min to max) showing that mechanically blocking muscle contraction by stretching, reduced GB number in Sqh-RNAi and control groups. Relaxed: Control n = 313 NMJs/50 larvae, Sqh-RNAi n = 187 NMJs/40 larvae (as in Fig. 5b), Stretched: Control n = 81 NMJs/15 larvae, Sqh-RNAi n = 71 NMJs/15 larvae, g Histogram showing relative frequency (%) of GBs in each class for relaxed or stretched animals. Statistical significance determined using non-parametric Kruskal-Wallis test (two-tailed); ****p < 0.0001, **p < 0.01, ns is not significant. Boxplots show 25^th–75^th percentiles, line at median, and whiskers from minimum to maximum. All data points are represented. Source data provided as a Source Data file.