The pool of synaptic vesicles in a nerve terminal is only sufficient for a few seconds worth of intense synaptic activity. To allow synaptic activity to continue, a highly efficient mechanism for synaptic vesicle biogenesis in nerve terminals is triggered by synaptic vesicle exocytosis. At maximum capacity, the recycling machinery can keep up with prolonged release rates in the range of 5-10 Hz. Although several proteins are believed to contribute to the recycling mechanism only one, dynamin, has been identified genetically as a participant. Temperature sensitivity mutations in dynamin give rise to the shibire class of temperature-sensitive paralytics in Drosophila melanogaster. We will use the wild type and mutant dynamins in a search for proteins that interact with dynamin. We will explore, using biochemical and morphological techniques, what binds dynamin on to recycling membranes and what causes it to be released. To explore the function of the proteins we isolate, we examine their ability to regulate the binding of dynamin to membranes in vitro. We also express mutant forms of dynamin, defective in protein association, in transgenic flies, both wild type and carrying temperature-sensitive shibire allele. Because of the importance of vesicle recycling to synaptic function, long term presynaptic changes in synaptic efficiency could involve changes in the amounts or post- translational modification of dynamin and its associated proteins.
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