Extended Data Fig. 6: Enrichment of extracellular vesicle machinery in post-synaptic apparatus of NMJs.

a) Representative images for the localization of CD63 (upper panel), CD81 (mid panel) and CHMP2A (lower panel) in healthy GC muscle NMJs. Blue indicates NFH, red indicates BTX, Dynamic blue-green-yellow indicates CD63/CD81/CHMP2A according to raw fluorescent intensity (blue=low; yellow=high). Scale bar: 10 µm. Experiment repeated 3 times (CD63, CD81) and 6 times for CHMP2A. b) Representative images for the localization of CD63 (upper panel), CD81 (lower panel) in NMJs within dissociated muscle fibers following enzymatic digestion of surrounding cells and connective tissues. Red indicates BTX, Dynamic blue-green-yellow indicates CD63/CD81 according to raw fluorescent intensity (blue=low; yellow=high). Scale bar: 10 µm. Experiment repeated 3 times. c) Quantitative analysis of CD81 raw fluorescent signal in synaptic versus extrasynaptic regions within the same muscle fiber. Two-tail paired student’s t-test. ****p = 7.68 × 10⁻⁵. n = 20 muscle fibers from 3 mice d) Representative images and 3D Imaris puncta colocalization analysis of extracellular vesicle machinery in postsynaptic apparatus of enzymatically denervated EDL muscle. White indicates BTX, yellow indicates CD-63, magenta indicates CD-81. Imaris puncta analysis was performed on BTX colocalization channels with CD-63 and CD-81. Scale bar: 10 µm. Experiment performed once. e) Representative max intensity projected (MIP) low magnification images (upper panel) and high magnification images of BTX-positive region (lower panel) of CD63-pHluorin signal in cultured primary muscle before (pre) and immediately after application of ammonium chloride (NH₄Cl). Dynamic magenta-orange-white indicates CD63-pHluorin intensity in low magnification images. Lower panel: magenta indicates BTX, green indicates CD63-pHluorin signal. Scale bars: 50 µm; 10 µm in upper and lower panels respectively. Experiment repeated in 2 neuromuscular co-cultures.