The subsynaptic network of MTs at the neuromuscular junction is induced by agrin. (a) NMJ in mouse sternomastoid muscle labeled for AChRs (red) and for Tyr-tubulin (green) reveals subsynaptic network of MTs. Bar, 10 µm. (b) MTs at the NMJ are posttranslationally modified by de-tyrosination. Labeling for β-tubulin, Glu-tubulin, and AChRs reveals synaptic and nonsynaptic MTs (red), selective de-tyrosination of subsynaptic MTs (green), and AChRs (blue). Note MTs are also present in presynaptic nerve terminal branches (arrowheads). Bar, 10 µm. (c) Network of MTs (tyr-tubulin, green) is present at nerve-free, ectopic postsynaptic membranes (marked by accumulation of AChRs [red]) induced by ectopic application of recombinant neural agrin to nonsynaptic region of adult muscle fiber. Note the accumulation of muscle nuclei, revealed by the surrounding MTs, at same site. Bar, 20 µm. (d) Tips of growing MTs are enriched at synaptic AChR clusters in epitrochleo-anconeus (ETA) muscle from GFP-Clip-170^ki/ki mutant mouse. Top: en-face view of NMJ marked by AChRs (red). MT plus-ends are stained with anti-GFP antibody. Bar, 5 µm. Bottom: part of a synaptic AChR cluster (red) stained for CLIP-170 with anti-GFP antibody (green) and reconstructed in 3D from a stack of confocal images. Left, view from muscle side of AChR cluster; note the MT plus-ends approaching the synaptic AChR cluster from below the postsynaptic membrane. Right, view from presynaptic side of same AChR cluster; note absence of MT tips from primary synaptic gutter where nerve terminal was located (not stained).