Neurotransmission requires the fusion of synaptic vesicles with the plasma membrane followed by the rapid recycling of vesicle membrane and protein components of the synaptic vesicle. There are three essential steps to recycling a synaptic vesicle. First, the area of the membrane to be recycled is pulled into the nerve terminal by formation of a clathrin protein coat, a process mediated by the clathrin adaptor complex AP-2. Next, the GTPase dynamin cleaves the neck of the vesicle, releasing it into the cell. Finally, the clathrin coat is rapidly removed so the vesicle can fuse with other membrane compartments.

Previously, we demonstrated that unc-26 encodes a protein with a high degree of identity to the vertebrate lipid phosphatase synaptojanin. Synaptojanin converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-4-phosphate (PIP). Two observations suggest that synaptojanin may play a role in synaptic vesicle recycling. First, it binds to proteins implicated in vesicle recycling and second, these proteins bind PIP2, the substrate of synaptojanin.

To demonstrate a role for synaptojanin in vesicle recycling, we conducted a detailed ultrastructural analysis of unc-26(s1710), our best candidate for a molecular null. In the absence of synaptojanin, we see several recycling defects. First, there is a general depletion of vesicles at the nerve terminal, a phenotype characteristic of endocytosis mutants. Second, membrane invaginations, both coated and uncoated, are seen at the nerve terminal. These structures may represent a block at a late step in vesicle budding, just prior to cleavage. Finally, we see vesicles which have been released into the cell, but still maintain their protein coat, suggesting that the lipid composition of the membrane is critical for vesicle uncoating. In addition to these vesicle recycling defects, we see other unexpected defects in the two major cytoskeletal elements, actin and tubulin. Based on these observations, we hypothesize that the accumulation of PIP2 in unc-26 mutants disrupts the distribution of proteins that mediate various steps of the recycling pathway, as well as altering cytoskeletal dynamics. To test this hypothesis, we are examining the distribution of PIP2 in unc-26 mutants, as well as looking at the distribution of endocytic proteins in an unc-26 background.