| Summary: | Synapse formation begins with the recognition of appropriate targets and formation of incipient contacts, and culminates with the recruitment of pre- and postsynaptic proteins to points of cell-cell contact. It is still unclear how cell-cell contact translates into the recruitment of synaptic proteins. Previous studies have shown that the cadherin/β-catenin cell adhesion complex plays an important role in localizing synaptic vesicles to developing synapses. This dissertation discusses work elucidating the transduction pathway that is activated following cell-cell contact, leading to the recruitment and retention of synaptic vesicles to presynaptic compartments. Using rat and mouse primary hippocampal cultures as a model system, we have demonstrated that β-catenin mediates the localization of synaptic vesicles to synapses through its recruitment of the PDZ scaffold protein, scribble, and it’s subsequent recruitment of the Rac/Cdc42 guanine nucleotide exchange factor, β-pix. We further demonstrate that β-pix enhances actin polymerization at these discrete sites, which is important for the “trapping” of synaptic vesicles as they translocate along the axon. We have demonstrated that cadherin, β-catenin, scribble, and β-pix form a complex at developing synapses using immunohistochemistry coupled with immunoprecipitation assays using synaptosomal fractions. Knockdown of β-catenin, scribble or β-pix using RNA interference (RNAi) disrupts the appropriate localization of synaptic vesicles at synapses. We have ordered this pathway and have shown that β-catenin is important for the recruitment and clustering of scribble to synapses, but that scribble knockdown does not affect β-catenin localization. We have also demonstrated that scribble knockdown disrupts the clustering of β-pix at synaptic sites. This complex has shown to control vesicle localization through β-pix-mediated enhancement of actin polymerization at these discrete sites. Indeed, β-pix knockdown results in decreased actin polymerization at synapses. Importantly, restoring actin polymerization at synapses through cortactin overexpression rescues the mislocalization of synaptic vesicles. This work provides novel insights into the molecular and cellular mechanisms underlying the development presynaptic compartments. === Medicine, Faculty of === Graduate
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