The regulation of dynamin I interactions in synaptic vesicle endocytosis

Abstract

Synaptic vesicle endocytosis is essential for the maintenance of synaptic transmission. The process involves a cascade of protein-protein interactions involving many proteins, the majority of which are essential. This pathway was investigated by perturbation of protein-protein interactions using the penetratin vector system. This thesis shows that penetratin tagging of peptides is a powerful tool to allow translocation ofpeptides across the lipid bilayer of isolated nerve terminals where the effect on synaptic vesicle turnover was monitored using styryl dyes. Depending on the target sequence the peptides were able to specifically inhibit either synaptic vesicle exocytosis or endocytosis.Dynamin I is a 96kDa GTPase and is one of eight proteins known to undergo coordinated dephosphorylation upon nerve terminal stimulation and subsequent rephosphorylation. They are collectively referred to as the dephosphins and are all dephosphorylated by calcineurin and at least three are rephosphorylated by cyclindependent kinase 5. Phosphomimetic peptides were used to investigate the specific role of dynamin I phosphorylation in synaptic vesicle endocytosis. The peptide mimicking dephosphorylated dynamin I was nearly four times more effective at inhibiting synaptic vesicle endocytosis than the peptide mimicking phosphorylated dynamin I, indicating that there is an essential role for these phosphorylation sites in endocytosis. Experiments to probe the function of the phosphoryation sites identified syndapin I as a stimulation dependent binding partner to this region of dynamin I. The interaction was further characterised by immunoprecipitation and GST-pulldown assays.Dynamin I can also be regulated by Ca²⁺ either directly or indirectly through Ca²⁺- dependent binding partners. Dynamin I has been shown to bind to the cytosolic C-terminus of the integral vesicle protein synaptophysin in a Ca²⁺ -dependent manner. This thesis shows that the over-expression of the C-terminus of synaptophysin-eGFP in cerebellar granule neurons inhibits synaptic vesicle recycling and that a peptide designed from this region of synaptophysin also inhibits synaptic vesicle recycling in isolated nerve terminals. GST-pulldowns with the C-terminus of synaptophysin extracted dynamin I, in the presence of Ca²⁺, but also a more prominent band shown by western blot to be amphiphysin I. Amphiphysin I was confirmed to bind to the Cterminus of synaptophysin independently from dynamin I through the use of a peptide which disrupts binding of dynamin I and amphiphysin I and through an in vitro binding assay. This suggests that amphiphysin I, as well as dynamin I, may interact with synaptophysin in nerve terminals, linking synaptophysin to a potential role in synaptic vesicle endocytosis.