Photoinactivation of Dynamin results in massive bulk membrane uptake, whereas shits1 mutant boutons at restrictive temperature show an accumulation of invaginated pits. (A and B) Electron micrographs of yw (A) and shi12-12B; shi-4C (B) control boutons stimulated for 5 min with KCl without FALI. Arrows, T bar; m, mitochondria. (C–H) Electron micrographs of shi12-12B; shi-4C boutons in which Dynamin was photoinactivated using FALI and subsequently stimulated for 5 min with KCl. High magnifications of the membrane with an active zone decorated with a T bar (E) and of inner membrane inclusions (F–H). Arrowheads, submembrane; arrow, T bar; m, mitochondria. (I) Cumulative probability distributions of vesicular profile diameter size in yw (n = 1,291 vesicles from three larvae), shi12-12B; shi-4C controls not treated with FALI (n = 1,291 vesicles from three larvae), and shi12-12B; shi-4C treated with FALI (n = 2,824 vesicles from three larvae). (J–L) Quantification of different boutonic features: the number of synaptic vesicles with a diameter <80 nm per area (J), the number of synaptic vesicles with a diameter >80 nm per area (K), and the number of invaginated pits per area (L) in yw controls (n = 33 bouton profiles from three larvae), shi12-12B; shi-4C after FALI (n = 27 bouton profiles from three larvae), and shits1 at a restrictive temperature (33°C; n = 16 profiles from three larvae). Error bars show SEMs; ANOVA (post hoc Tukey’s test): ***, P < 0.0001. (M and N) Electron micrographs of shits1 boutons stimulated for 5 min with KCl at permissive (25°C; M) and restrictive (33°C) temperature (N). Asterisks, invaginated pits; arrow, T bar; m, mitochondria. (O–Q) Higher magnification of the active zones in shits1 boutons stimulated for 5 min with KCl at permissive (O) and restrictive temperature (P) and of invaginated pits in shits1 boutons at restrictive temperature (Q). Note the lack of synaptic vesicles around the active zone in shits1 at restrictive temperature. Bars: (A–H, M, and N) 0.5 µm; (O–Q) 0.1 µm.
