Genetic analysis of endocytosis at the Drosophila synapse

Abstract

Endocytosis plays an essential role in maintaining a pool of synaptic vesicles for sustained neurotransmitter release. Synaptic vesicles are internalized and fuse with endosomes, and are subsequently reassembled to be ready for another round of exocytosis. Here I describe in two distinct studies the function of endosomes at synapses and regulation of dynamin, a protein essential for endocytosis, using the Drosophila synapse as a model. To study the function of endosomes at synapses I analyzed the localization and function of two Drosophila endosomal proteins, Hook and Deep orange (Dor), at the larval neuromuscular junction. I present here the first genetic evidence of a role for endocytic trafficking in plasticity of the synapse. I also found that mutations in hook and dor affect the number of varicosities at the nerve terminal without affecting synaptic vesicle recycling, indicating that Hook and Dor proteins play a role in later stages of endocytosis at the synapse. Dynamin is a GTPase that is essential for internalization of synaptic vesicles from the plasma membrane. Flies carrying shi ts mutations have a conditional defect in dynamin function. Molecules that regulate GTP loading (guanine-nucleotide exchange factors-GEFs) and GTPase activity (GTPase activating proteins-GAPs) of dynamin are unknown. Here I describe the identification of such molecules/domains by analyses of enhancer and suppressor mutations identified in previously conducted genetic screens. I show here that the enzymatic activity of Nucleoside diphosphate kinase (NDP Kinase), a source of GTP encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, is essential for dynamin function at synapses. Dynamin is also regulated by an intramolecular GTPase effector domain (GED) and I have identified separate mutations in shi, which map to the GED, that suppress endocytic defects in shits2. Overall, these data indicate a model in which the stability of dynamin: GTP is opposingly regulated by an unusual GEF activity of NDP kinase and a GAP activity in dynamin; in addition these findings indicate the possibility of an intriguing therapy for nm23 tumor progression.