Our long-term objectives are to determine the sub-cellular processes, and their molecular substrates, which control the efficiency of synaptic transmission in the central nervous system (CNS). Our approach is to use methods that allow us to isolate and study distinct steps in the cycling of synaptic vesicles in the presynaptic terminal. Our previous work demonstrates the usefulness and applicability of a number of different optical probes, including the fluorescent tracer FM 1-43 and the more recently-developed pH-based sensor of synaptic activity, synapto-pHIuorin, for studying synaptic vesicle recycling in single presynaptic terminals in dissociated neuronal cell cultures. Here we propose to examine several aspects of the coupling of exocytosis and endocytosis and how it is controlled using biophysical, molecular and genetic means. Our working hypothesis is that the proper functioning of the complete synaptic vesicle cycle is critical to coordinating information flow in the brain. Because of its cyclic nature, modulation at any point in the synaptic vesicle cycle could prove important in controlling synaptic efficacy. Similarly, dysfunction of any of the steps in the vesicle cycle, such as might arise in specific mutations in diseased states, could lead to impairment of synaptic transmission. One of the critical steps in the synaptic vesicle cycle is the endocytic retrieval of synaptic vesicle membrane and proteins for future reuse. We propose 4 specific aims to examine how endocytosis is controlled and how different molecules participate in endocytosis for different types of physiological stimuli. ? 1) Examine the role of dynamin-1 in synaptic vesicle endocytosis at CNS nerve terminals ? 2) Examine the role of synaptojanin-1 in synaptic vesicle endocytosis at CNS nerve terminals ? 3) Examine the role of clathrin in synaptic vesicle endocytosis at CNS nerve terminals ? 4) Examine the role of different endocytic adaptors as well as the importance of sorting motifs and interactions with synaptophysin in controlling synaptic vesicle membrane protein recapture and endocytosis. ? ?
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