Fig. 5: Interfering with MyoII alters MNs capacity to form GBs.
From: Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction
a Representative images of control and MyoII K/D animals with (left) or without (right) high-K+ stimulation. We used NSyb-Gal4 to express RNAi against the two subunits of MyoII (Spaghetti squash – Sqh, regulatory light chain, and Zipper – Zip, heavy chain) in neurons. Presynaptic and postsynaptic membranes labeled with antibodies for Horseradish peroxidase (HRP, gray) and Discs-large (Dlg, green), respectively; ghost boutons (GBs) identified by white arrows. Scale bar, 10 µm. b Boxplot (min to max) showing that neuronal RNAi against Sqh and Zip increased GB frequency after stimulation. c Histogram showing relative frequency of GBs (%) in each class for the stimulated NMJs. Control n = 313NMJs/50 larvae, Sqh-RNAi n = 187NMJs/40 larvae, Zip-RNAi n = 69NMJs/20 larvae. d Boxplot (min to max) showing that neuronal RNAi against Sqh and Zip did not change GB frequency during development (unstimulated control). e Histogram showing frequency of GBs (%) in each class for unstimulated NMJs. Control n = 85NMJs/22 larvae, Sqh-RNAi n = 56NMJs/14 larvae, Zip-RNAi n = 47NMJs/14 larvae f Time-lapse images of bouton formation when MyoII was K/D in neurons. NSyb-Gal4 used to drive UAS-CD4-Tom (membrane label) and UAS-Sqh-RNAi expression. Scale bar, 2 µm. Arrows indicate where boutons emerge. N = 13 NMJs/larvae (control), 5 NMJs/larvae (Sqh-RNAi). g Boxplot (min to max) reduced bouton formation time in neuronal MyoII K/D. h Boxplot (min to max) showing that GB area remained unaltered in neuronal MyoII K/D. N = 13 larvae/46 boutons (for control), 5 larvae/46 boutons (for Sqh-RNAi). Statistical significance determined using non-parametric two-tailed tests: Kruskal–Wallis (b, d) or Mann–Whitney (g, h); *p < 0.05 (p = 0.0254), ****p < 0.0001, ns not significant. Boxplots show 25th–75th percentiles, lines at the median, and whiskers from minimum to maximum. All data points are represented. Plots with linear regression between time and area for driver control (i) and Sqh-RNAi (j). 95% confidence bands of the best-fit line are indicated by dotted lines. R², Spearman’s correlation value (rs), and respective p values, are shown in the graphs. k Schematics of the factors that promote bleb formation. l 3-D schematic of muscle contraction effect on neuronal MyoII K/D. Source data provided as a Source Data file.
