The Role of amphiphysin in synaptic vesicle endocytosis. B.M. Chow , P.A. Leventis , B.A. Stewart , G.L. Boulianne. Hospital for Sick Children, Toronto, Ontario, Canada.

   First discovered in association with synaptic vesicles (SVs) in chick brain, amphiphysin (amp) is believed to be important in SV endocytosis. Biochemical assays have demonstrated that amp binds to proteins involved in SV endocytosis, such as dynamin and clathrin. One model suggests that amp targets proteins to clathrin-coated vesicles. However, amp function in vivo is still unknown. We propose to define amphiphysin's role in endocytosis in vivo using Drosophila as a model organism. Molecular characterization of Drosophila amphiphysin (Damp) reveals multiple isoforms of the protein that are highly related to the vertebrate homologue at the N and C termini. However, consensus sequences for binding to other endocytic proteins, such as clathrin, are absent. To study Damp function, we have taken several approaches, such as the production of antibodies for biochemical studies, and the generation of mutant and transgenic flies. Using an antibody generated against full-length Damp, we have found that several Damp isoforms are widely expressed throughout development. Damp is also expressed in a broad range of tissues, including the CNS, discs and body wall. Studies are currently underway to determine Damp protein-protein interactions in vitro. To explore Damp function in vivo, we have generated mutant and transgenic flies. Surprisingly, null mutants are viable, but sluggish, and show no defects in synaptic morphology or synaptic physiology, suggesting that Damp is not critical for SV endocytosis. We are currently characterizing Damp transgenic flies, which overexpress truncated Damp constructs under the control of the GAL4-UAS system. These constructs should act in a dominant-negative manner to suppress Damp function. We are now testing these flies with different GAL4 drivers to find a phenotype that will be useful for second site modifier screens. The discovery of novel protein partners for Damp may reveal functions for Damp beyond its putative role at the synapse.