Dynamin photoinactivation blocks Clathrin and α-adaptin recruitment and induces bulk membrane retrieval

Dynamin is a well-known regulator of synaptic endocytosis. Temperature-sensitive dynamin (shits1) mutations in Drosophila melanogaster or deletion of some of the mammalian Dynamins causes the accumulation of invaginated endocytic pits at synapses, sometimes also on bulk endosomes, indicating impaired membrane scission. However, complete loss of dynamin function has not been studied in neurons in vivo, and whether Dynamin acts in different aspects of synaptic vesicle formation remains enigmatic. We used acute photoinactivation and found that loss of Dynamin function blocked membrane recycling and caused the buildup of huge membrane-connected cisternae, in contrast to the invaginated pits that accumulate in shits1 mutants. Moreover, photoinactivation of Dynamin in shits1 animals converted these pits into bulk cisternae. Bulk membrane retrieval has also been seen upon Clathrin photoinactivation, and superresolution imaging indicated that acute Dynamin photoinactivation blocked Clathrin and alpha-adaptin relocalization to synaptic membranes upon nerve stimulation. Hence, our data indicate that Dynamin is critically involved in the stabilization of Clathrin-and AP2-dependent endocytic pits.