Sustained neurotransmission requires the recycling of synaptic vesicle components from the plasma membrane via endocytosis to restore the pool of synaptic vesicles in the presynaptic nerve terminal. We have taken two approaches to study synaptic vesicle endocytosis in C. elegans. First, we are cloning synaptic function mutants and characterizing their phenotypes. Second, we are identifying worm homologs of vertebrate endocytic proteins, then looking for exisiting mutations or screening for new mutations in these genes. Currently, we are cloning ric-1 and unc-70, and we have identified two worm homologs of synaptojanin, an inositol-5-phosphatase.

Two observations suggest that ric-1 is likely to be a presynaptic defect: 1) ric-1 homozygotes are resistant to inhibitors of cholinesterase; and 2) homozygotes exhibit the same jerky, uncoordinated, coiler phenotypes as animals lacking acetylcholine. We have mapped ric-1 to a cosmid gap within the mec-14 - lin-39 interval on LGIII. We are now cloning transposon insertions associated with spontaneous alleles (kindly provided by Jim Rand) isolated from the strain TR638 which displays high levels of transpogenesis.

Dominant alleles of unc-70 have a curly posture and are hypersensitive to inhibitors of cholinesterase, suggesting that such mutants release elevated levels of acetylcholine. Recessive alleles of unc-70 are lethal, but escapers are resistant to inhibitors of acetylcholinesterase indicating that these mutants reduce the levels of secreted acetylcholine. Thus, the level of UNC-70 activity seems to regulate the level of neurotransmitter released. We have rescued unc-70 null alleles by microinjection and these rescued animals have a curly phenotype that resembles dominant alleles of unc-70.

Increasing evidence suggests a link between phosphoinositide metabolism and endocytosis, including the requirement of PI-3 kinase for efficient growth receptor internalization and downregulation, via a dynamin/clathrin mediated-process. Recently, a neuronal-specific inositol-5-phosphatase, dubbed synaptojanin, has been identified in rats. Synaptojanin interacts with amphyphysin and the adaptor protein GRB2. The only other protein currently known to interact with amphyphysin is dynamin. Synaptojanin colocalizes with dynamin at the nerve terminal, suggesting a role for synaptojanin in endocytosis. In conjunction with our collaborators, Pietro deCamilli and Yasuo Nemoto, we have identified two loci homologous to rat synaptojanin. We are presently mapping the physical location of these genes.