Studies of membrane-binding proteins involved in synaptic vesicle recycling
Abstract: Signaling between neurons occurs at synapses. A presynaptic nerve terminal releases neurotransmitter, which diffuses over the synaptic cleft, and binds to postsynaptic receptors. Release occurs by fusion of neurotransmitter-containing vesicles with the plasma membrane. To support sustained transmitter release the synaptic vesicles are recycled locally at the nerve terminal. After the synaptic vesicle has collapsed into the plasma membrane synaptic vesicles can be retrieved by two different mechanisms, clathrin-mediated endocytosis and bulk endocytosis. Both recycling pathways depend on interplay between many proteins, several of which bind to and can rearrange lipid membranes. The aim with this thesis is to investigate the role of three different proteins; epsin, endophilin and syndapin, each containing a lipid binding domain known to induce membrane curvature in vitro, in synaptic vesicle recycling at a living synapse. The model system used is synapses located in the giant reticulospinal axons in lamprey that permits acute perturbation of protein-protein and protein-lipid interactions by micro-injection of reagents. These synapses have an organization that facilitates quantitative morphological analysis. Perturbation of each of the three different proteins caused a stimulus-dependent decrease in the number of synaptic vesicles in the synaptic vesicle cluster suggesting an involvement in synaptic vesicle recycling. Epsin appears to be involved in recruitment or assembly of the clathrin coat since perturbation of the membrane-binding ENTH domain of epsin resulted in a decrease of coated intermediates. In further support of this possibility, coated intermediates with distorted structure was observed after perturbation of the clathrin and AP2 binding region of epsin. Perturbation of the membrane-binding BAR domain of endophilin with IgG and Fab fragments resulted in accumulation of shallow coated pits at the plasma membrane. Injection of the BAR domain also resulted in accumulation of shallow coated pits. However, similar coated pits occurred after injection of an F-BAR domain with different curvature indicating that the curvature-inducing properties of the BAR domain is not directly involved in the progression of coated pit invagination. Therefore the accumulation of shallow coated pits is probably linked to the binding partners of endophilin. Clathrin-mediated endocytosis did not show any detectable change in synapses at which syndapin had been perturbed. Instead there was an accumulation of membranous cisternae containing synaptic vesicle membrane and with associated coated pits, indicating an involvement of syndapin in bulk endocytosis and/or stabilization of the plasma membrane at high rates of synaptic activity.
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