This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The overall goal of these studies is to elucidate the membrane trafficking pathway that is used in the recycling of synaptic vesicles (SVs) in living synapses. The collaboration with NCMIR involves high-resolution ultrastructural analysis of endocytic intermediates trapped after perturbation of protein-protein interactions in a living synapse. Giant reticulospinal synapse in lamprey serves as a model system for these experiments. It was demonstrated in our join experiments that compounds, which disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impaired synaptic vesicle endocytosis. Two distinct endocytic intermediates accumulated. 'Free' clathrin-coated vesicles were induced by a peptide blocking endophilin s SH3 domain (PP-19), and by antibodies to the proline-rich domain of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. These data indicate that the SH3 domain of endophilin participates in both fission and uncoating, and is a key component of a molecular switch, which couples the fission reaction to uncoating. Since 'free' coated vesicles were found at significant distances from active zones it was suggested that uncoating is necessary for the proper delivery of vesicles to the sites of release (Gad et al., 2000).
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