Endophilin is an SH3 domain-containing protein which binds the proline-rich domains of synaptojanin and dynamin. Both dynamin and synaptojanin play a major role in clathrin-mediated endocytosis. Recent studies on the Drosophila endophilin mutant, D-endoA/endo, demonstrated that mutant animals have a depletion of synaptic vesicles in nerve terminals, indicating a defect in vesicle recycling (1,2). Endophilin is encoded by the
unc-57 gene. We are characterizing
unc-57 mutants to determine 1) if endophilin is required for synaptic vesicle endocytosis in C. elegans, 2). if the role of endophilin is to bind and localize synaptojanin and/or dynamin to sites of endocytosis, and 3). if the lipid modifying activity of endophilin is required for its function. Consistent with a role in endocytosis,
unc-57 is expressed in many, if not all neurons and in the spermatheca. In addition, GFP-tagged UNC-57 protein is present at synapses. Physiological recordings demonstrate that
unc-57 mutants have reduced evoked and endogenous activity. This is consistent with a defect in either exocytosis or endocytosis. However, synapses fatigue more rapdily in mutants than wild-type animals. These data are consistent with endophilin playing a role in synaptic vesicle recycling. Moreover, proteins normally endocytosed into vesicles are diffusely distributed on neuronal processes. Specifically, a GFP-tagged synaptic vesicle protein, synaptobrevin (VAMP), is diffusely localized in the
unc-57(
ok310) mutant background rather than punctate as is seen in wild-type worms. This result is similar to the phenotype of other endocytosis mutants such as
unc-26,
dyn-1,
unc-11, and
snt-1. Finally, proof of an endocytosis defect will require an ultrastructural analysis; these experiments are in progress. It has been suggested that endophilin is required to bind and localize dynamin and synaptojanin to sites of endocytosis. To test this we are looking at the localization of DYN-1::GFP and UNC-26::GFP in an
unc-57 mutant background. Our data suggests that at a gross level, DYN-1:GFP is correctly localized in
unc-57 mutants. We do not yet know if UNC-26 is localized. Schmidt et al. recently showed that endophilin has a lipid modifying function - specifically, lysophosphatidic acid acyl transferase activity (LPAAT) (3). This lipid modifying activity converts lysophopatidic acid to phosphatidic acid. It was proposed that a specific lipid composition may be required for plasma membrane invagination during synaptic vesicle endocytosis. We are considering a variety of approaches to determine if C. elegans endophilin has LPAAT activity and if that activity is required for its role in synaptic vesicle endocytosis.