Coordination of endocytosis at the synaptic periactive zone

Abstract: Abstract: Neurons in the central nervous system communicate via specialized junctions called synapses. Neurotransmitter-filled vesicles are clustered at these junctions, where they are released in response to synaptic activity. To sustain reliable neurotransmission the synaptic vesicles are recycled locally. At least two recycling mechanisms, bulk and clathrin-mediated endocytosis, occur in a region adjacent the active zone, the periactive zone. Clathrin-mediated endocytosis is dependent on a number of protein-protein and protein-lipid interactions that drive clathrin polymerization, membrane invagination, and scission. The aim of this thesis is to investigate how compensatory endocytosis is coordinated in the synaptic periactive zone. The studies are focused on actin dynamics in the periactive zone, molecular adaptations of the endocytic machinery in synapses with different firing properties, recruitment of endocytic proteins to the periactive zone, and the molecular steps leading to fission of clathrin-coated intermediates. The following conclusions were drawn from the experiments presented in this thesis: ' Synaptic activity promotes actin filament formation in the periactive zone and dispersion of synapsin from the synaptic vesicle cluster. Perturbation of synapsin function leads to a disruption of actin filament formation. ' Endocytic proteins dynamin, amphiphysin, and intersectin redistribute from the synaptic vesicle cluster to the periactive zone in response to synaptic activity. ' Synapses established by axons with a tonic activity pattern have higher levels of synapsin, dynamin, amphiphysin, and intersectin compared to those with phasic activity. This may contribute to the resistance to fatigue displayed by tonic synapses. ' Dynamin, amphiphysin, and intersectin are recruited to initial stages during clathrincoated vesicle formation. ' Disruption of amphiphysin s interaction with coat components by antibodies results in formation of clathrin-coated pits with an abnormal shape. This suggests that amphiphysin is an integral component of the clathrin coat. ' Dynamin is recruited to clathrin-coated pits independently of intersectin. However, intersectin is important for the recruitment of dynamin to the fission complex. Taken together these results show that clathrin-mediated membrane retrieval in synapses require coordinated trafficking of endocytic proteins between active and periactive zones.