Synaptic vesicle endocytosis studied in a living synapse

Neurons in the central nervous system communicate by transmitting electrical signals at special sites of contact called synapses. Synapses contain neurotransmitter-filled synaptic vesicles, which fuse with the presynaptic plasma membrane. The neurotransmitter diffuses to the neighboring neuron and triggers a new electrical signal. In order to maintain synaptic transmission, the synaptic vesicle membrane must be retrieved from the presynaptic plasma membrane to be reused for another round of transmitter release. In one proposed model, the synaptic vesicles fuse completely with the plasma membrane and the vesicle membrane is retrieved through clathrin-coated buds. In another model, the synaptic vesicle connects transiently to the plasma membrane and clathrin is not involved in the membrane retrieval. Clathrin-mediated endocytosis depends on a number of accessory proteins that appear to assist the clathrin-coated intermediate at different steps in the process. The aim of this thesis was to investigate the role of clathrin-coated buds in synaptic vesicle retrieval. The function of the accessory proteins endophilin, dynamin, amphiphysin and synaptojanin in clathrin-mediated endocytosis was also examined. For this purpose, the giant reticulospinal synapse in lamprey was employed. The organization of this synapse allows acute perturbations of synaptic vesicle recycling. The following conclusions were drawn from the experiments presented in this thesis: • Impairment of several clathrin-associated proteins led to an inhibition of synaptic vesicle endocytosis and accumulation of clathrin coated intermediates. This suggests that the main pathway for synaptic vesicle retrieval is through clathrin-mediated endocytosis. • After temporally dissociating synaptic vesicle release from endocytosis, clathrin-mediated retrieval could be initiated by readding low micromolar concentrations of extracellular Ca, without additional action potential stimulation. The results indicate that the synaptic vesicle membrane incorporated in the plasma membrane during exocytosis is a sufficient trigger of synaptic vesicle endocytosis. • Antibody-mediated disruption of endophilin led to a massive accumulation of shallow clathrincoated pits on the presynaptic plasma membrane. This suggests that endophilin is required for the invagination of the shallow coated pit, possibly by altering the lipid composition of the coated membrane bud. • Microinjection of compounds that disrupt the binding of dynamin to amphiphysin and endophilin blocked clathrin-mediated retrieval at the stage of deeply invaginated coated buds with a constricted ‘neck’. These data indicate that dynamin and its interaction with amphiphysin and endophilin is essential in the fission of the neck of the coated pit. • Injection of a peptide blocking the endophilin-synaptojanin interaction and antibodies to the proline-rich domain of synaptojanin induced accumulation of free clathrin-coated vesicles. These results suggest that synaptojanin is recruited to the free coated vesicle by the interaction with endophilin to facilitate the removal of the clathrin coat. Endophilin may be a part of a molecular switch that couples the fission reaction to uncoating and imparts a strict vectorality to synaptic vesicle endocytosis. • Dynamin was shown to be associated with both early and late stages of coated pits. Antibodymediated disruption of dynamin blocked synaptic vesicle endocytosis at a stage preceeding clathrin coat formation. These results indicate that dynamin participates at early stages of endocytosis in addition to its role in fission. The results presented in this thesis have increased the understanding of how synaptic vesicles are retrieved and the molecular mechanisms that govern clathrin-mediated endocytosis, a process important in all animal cells to regulate their response to the external environment.