Fig. 3.

Mislocalization of dynamin from the periactive zone in the dap160 mutant (Δ1/Df). (A–C) Confocal microscopy images showing the localization of (A) dynamin (Dyn), (B) Eps15 and (C) AP2 at rest in control NMJs. (D–F) Following stimulation there is a redistribution of (D) dynamin-ir, (E) Eps15-ir and (F) AP2-ir in control boutons. (G,H) Confocal images of dap160 mutant NMJs showing that Dyn (red) and Eps15 (green) are largely colocalized at rest and have similar distribution (G). During stimulation, dynamin is no longer accumulated in spots, unlike Eps15 (H). (I,J) Confocal images showing AP2-ir (red) and Eps15-ir (green) in dap160 mutant nerve terminals at rest (I) and after stimulation (J). Note colocalization of both proteins in spots at the plasma membrane (arrow). (K,L) Low magnification electron micrographs illustrating the overall view of dynamin immunolabeling in dap160 mutant NMJs at rest. (M) High-magnification image showing dynamin-ir within the SV pool in dap160 mutant at rest. (N) Mistargeting of dynamin in the mutant NMJ upon stimulation. (O) High-magnification image of a labeled constricted pit (arrow) accumulated at the periactive zone following high K⁺ stimulation. (P,Q) Quantification of dynamin localization in the synaptic vesicle pool (rest) and at the PAZ (stim) in control and dap160 mutant. Bar graphs indicate mean + s.e.m. *P<0.05; ***P<0.001 using Student's t-test. T, T-bar; m, mitochondrion; sv, synaptic vesicle; az, active zone. Scale bars: 2 µm (A–J); 0.5 µm (K,L); 0.1 µm (M); 0.2 µm (N); 50 nm (O).