Photoinactivation of Dynamin converts invaginated pits in shi^ts1 mutants into bulk cisternae. (A and B) Quantification of the HA-Chc (A, n = 11 boutons from seven larvae each) and α-Ada (B, n = 7–10 boutons from six to seven larvae) labeling intensity in a line over the (largest) bouton diameter (and normalized to the length of the bouton diameter; see Materials and methods) in KCl-stimulated shi^ts1/Y; HA-chc/+ at permissive (RT) temperature and shi^ts1/Y; HA-chc/+ at restrictive temperature (33°C); see Materials and methods. Averages are in black lines, and SEMs are in gray shades. rel., relative. (C) Model of the distribution of different Dynamin molecules (Shi-4C and Shi^ts1) in endocytic pits (based on anti-Dynamin labeling intensity—see Stimulus-dependent Clathrin and α-Ada recruitment in shi^ts1 mutants in the Results section) and calculation of the probability that Dynamin rings consist only of Shi^ts1 or only of Shi-4C in shi^ts1/Y; shi-4C/+ larvae. (D–I) FM 1-43 labeling in shi^ts1/Y; shi-4C/+ larvae treated (+; F–I) or not treated (−; D and E) with FlAsH for 10 min and illuminated for 2 min (+; G and I) at permissive (D, F, and G) or restrictive (E, H, and I) temperature. All preparations were stimulated for 5 min with KCl in the presence of FM 1-43. Note that shi^ts1/Y; shi-4C/+ animals at restrictive temperature without FALI phenocopy shi^ts1 animals and do not internalize FM 1-43, whereas shi^ts1/Y; shi-4C/+ animals at restrictive temperature after FALI also internalize FM 1-43 in cisternal inclusions, similar to shi^12-12B; shi-4C animals in which Dynamin was photoinactivated. Bar, 5 µm.