UBC Theses and Dissertations
Trafficking of scaffolding and adhesion proteins : the role of pre-assembled complexes and lateral diffusion during synapse development Gerrow, Kimberly A.
Part One: Role of a pre-formed scaffolding complex in excitatory synapse formation. In order to determine the role of non-synaptic clusters of postsynaptic proteins we monitored the trafficking of several candidate proteins implicated in synaptogenesis, when non-synaptic clusters of scaffold proteins are most abundant. We found a protein complex consisting of two populations that differ in their content, mobility, and involvement in synapse formation. One subpopulation is mobile and relies on actin transport for delivery to nascent and existing synapses. These mobile clusters contain the scaffolding proteins PSD-95, GKAP, and Shank. The second group consists of stationary non-synaptic scaffold complexes that mainly contain neuroligin-1, can recruit synaptophysin-containing axonal transport vesicles, and are readily transformed to functional presynaptic contacts that recycle the vital dye FM 4-64. These results postulate a mechanism whereby preformed scaffold protein complexes serve as predetermined postsynaptic hotspots for establishment of new functional excitatory synapses. Part Two: Neuroligin trafficking in live neurons. The mechanisms that govern the differential trafficking and retention of neuroligin-1 to glutamatergic synapses and neuroligin-2 to GABAergic synapses remain unclear. In order to monitor the recruitment/retention of neuroligin-1 and -2 to synaptic sites, a site-specific biotinylation-based approach was utilized that allows for the visualization of surface proteins in live neurons with monovalent streptavidin. To quantify these changes, FRAP (fluorescence recovery after photo beaching) showed similar recovery rates for GFP-tagged neuroligins (representative of the total pool) compared to AP-tagged neuroligins (representative of the surface pool). The mobile pool of neuroligin-1 clusters was significantly larger than neuroligin-2 clusters and was depressed in older neurons. The mobility of neuroligin-1 clusters was influenced by the expression of specific scaffolding proteins. Exogenous expression of PSD-95 or S-SCAM, but not Shank, reduced the mobile faction of neuroligin-1, whereas the mobile fraction of neuroligin-2 was reduced by exogenous expression of S-SCAM. Interruption of actin and microtubule cytoskeleton interactions also decreased the recovery of both neuroligin-1 and -2. Thus the differential mobility of neuroligin molecules as well as association with particular scaffolding proteins and the cytoskeleton may contribute to their lateral diffusion and retention at particular synaptic sites.
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