Impairment of Ank2 binding of Nrg180 increases NMJ growth.

<p>Analysis of NMJ growth in <i>nrg¹⁴</i> mutant animals expressing different mutated <i>nrg</i> Pacman constructs using the presynaptic vesicle marker Synapsin (Syn, green), the postsynaptic marker Dlg (red), and a marker for the presynaptic membrane (Hrp, blue). (A) Presence of the <i>nrg</i> Pacman wild-type construct resulted in wild-type muscle 4 NMJs. The inset shows individual presynaptic boutons at higher magnification. (B) The Nrg180^Y-F mutation resulted only in small alterations of NMJ growth. (C) The Nrg180^Y-A mutation led to a significant increase in NMJ length. (D) Deletion of the Nrg180–FIGQY motif resulted in a significant, almost 2-fold overgrowth and a corresponding reduction in the area of individual boutons. (E and F) Quantification of bouton number, NMJ length, and bouton area. NMJ growth defects correlated with an increasing loss of Ank2 binding capacities. No alterations were observed for mutations affecting the PDZ protein binding site of Nrg180 or the Nrg167–FIGQY motif. Values were normalized to wild-type rescue. Asterisks indicate highly significant changes (<i>p</i>≤0.001) for bouton number in (E) and for bouton area in (F) (<i>n</i> = 69–176 NMJs for bouton number, <i>n</i> = 20 NMJs for NMJ length, and <i>n</i> = 10 NMJs for bouton area quantifications). Scale bar in (A) corresponds to (A–D), 10 µm, inset 5 µm. Error bars represent SEM.