Photoinactivation of Dynamin blocks synaptic vesicle recycling. (A) Genomic dynamin construct tagged in the middle domain with a Flag-tetracysteine tag (shi-4C). PH, Pleckstrin homology; PRD, Proline-rich domain; GED, GTPase effector domain. (B–D) FlAsH fluorescence after incubation of yw controls (B) and shi^12-12B; shi-4C (C) third instar fillets in FlAsH reagent shows labeling only in boutons of animals expressing Shi-4C (C). (D) Anti-Dynamin (Dyn) labeling in yw animals. Bars, 20 µm. (E) Sample EJC traces recorded from muscle 6 in 0.5 mM of extracellular CaCl₂ in yw controls and shi^12-12B; shi-4C animals that were not subjected to FALI (−) and shi^12-12B; shi-4C after FALI (+). (F) Quantification of the EJC amplitude recorded in 0.5 and 2 mM CaCl₂ in controls yw and shi^12-12B; shi-4C without (−) and with FALI (+). Error bars show SEMs; ANOVA (post hoc Tukey’s test). n for 0.5 mM CaCl₂ = 7, 7, and 10 and for 2 mM CaCl₂ = 8, 7, and 5 recordings from four to nine larvae. (G) Cumulative probability histogram of miniature EJC amplitudes measured from yw controls and shi^12-12B; shi-4C incubated with FlAsH before illumination, during light inactivation and after FALI. n = 8, 5, 5, and 5 recordings from as many larvae. (H) Relative EJP amplitude measured during 10 min of 10-Hz stimulation in controls yw (n = 8 recordings from eight larvae) and in shi^12-12B; shi-4C loaded with FlAsH (n = 5 recordings from five larvae). Recordings were made by measuring EJPs for 2 min without illuminating the samples followed by 2 min of illumination to photoinactivate Dynamin. EJP amplitudes are plotted as the means of 30 s of recording and normalized to the means of the first 15 s per genotype. (inset) Example EJP data traces of yw and shi^12-12B; shi-4C (in black). Error bars show SEMs.