| Synaptic vesicles are reformed following neurotransmitter release through the process of endocytosis in nerve terminals. Local recycling results in rapid replenishment of synaptic vesicles and allows for continued synaptic activity in response to repeated stimuli. Although it is well established that synaptic vesicles are made through endocytic processes, the molecular mechanisms that form the vesicles are poorly understood. In fact, three complementary pathways have been proposed, each with its own use and mechanism. In this thesis, I have conducted cell biological and biochemical studies into the mechanisms of two of these pathways, formation of synaptic vesicles through an endosomal intermediate and formation of synaptic vesicles through direct budding from the plasma membrane.; Neurons have, at their nerve terminals, dynamic endosomal structures that are thought to be intermediates in synaptic vesicle biogenesis. In the PC12 neuronal model cell line, an arf1/AP-3-dependent endosomal budding mechanism is the predominant route of synaptic vesicle-like vesicle formation. This pathway is sensitive to reduced temperature and to the fungal metabolite, brefeldin A. I asked whether neurons use this same mechanism for synaptic vesicle formation by setting up an antibody uptake assay for synaptic vesicle biogenesis in cultured neurons. Using the assay, I found that synaptic vesicle formation is not sensitive to either reduced temperature or brefeldin A, thus demonstrating that the arf1/AP-3-dependent budding mechanism is not the primary mechanism of synaptic vesicle biogenesis in neurons.; Neurons can form synaptic vesicles directly from the plasma membrane using a clathrin-mediated budding mechanism. I characterized the activity of the clathrin accessory protein, intersectin 1L, a neuron-specific protein that contains a guanine exchange factor DH domain. Using recombinant proteins, I found that the guanine exchange activity is specific for Cdc42. The upstream SH3 domains inhibit exchange activity by direct association with the DH domain, thereby blocking Cdc42 binding and exchange. Interaction with SH3 domain binding partners was not found to release this inhibition. However, SH3 domain binding partners from divergent pathways do compete with each other to bind the SH3 domains suggesting regulation of actin polymerization in response to synaptic vesicle endocytosis. |
