Microtubule density is inversely proportional to Sec8 expression. A: Confocal images showing a portion of a NMJ on ventral longitudinal muscles 6 & 7 in third instar larvae (110–120 h AEL), visualized using antibodies against tubulin (green) and the essential glutamate receptor subunit GluRIIC (red). Upper left half of the image shows confocal projection; lower right half of the image shows 3D isosurface reconstruction of the same data. This shows the close association between pre and postsynaptic microtubules and the NMJ. B: Microtubules in postsynaptic larval muscles. Muscle nuclei ('N') are surrounded by microtubule networks (labeled with anti-acetylated tubulin antibody, magenta) that often extend (arrows) toward the neuromuscular junction (labeled with anti-HRP, green). This suggests that muscle microtubules may be critical for delivery of postsynaptic molecules to the NMJ. C: Quantification of synaptic microtubule density. To measure synaptic microtubule density, immunofluorescence from only the non-axonal area delimited by HRP staining (magenta) was measured. D: Quantification of extrasynaptic microtubule density. To measure extrasynaptic microtubule density, immunofluorescence from the muscle area not delimited by HRP staining was measured. E: Confocal images showing intersegmental nerve branch b (ISNb) innervating ventral longitudinal muscles in first instar larvae (24–28 h AEL). Neuronal tissue was visualized using anti-HRP antibody (magenta). Microtubules were visualized using an anti-acetylated tubulin antibody, which recognizes only stably polymerized alpha and beta tubulin (green). Individual NMJs are identified (arrows, labels). In 24B Gal4;UAS-sec8 animals, sec8 cDNA is overexpressed in postsynaptic muscle cells, resulting in small but not statistically significant reductions in microtubule immunofluorescence. In sec8 mutants, microtubule immunofluorescence is increased. This increase is highest at the synapse, where Sec8 is normally localized. Qualitatively identical results were obtained using another antibody that recognizes all tubulin (not shown). Scale bar: 10 μm.