Molecular mechanisms controlling dynamin recruitment to sites of endocytosis by SH3-domain containing proteins

Abstract

Endocytosis is a general cellular mechanism, which regulates a number of important events, including recycling of synaptic vesicles. Clathrin-mediated endocytosis is the best-characterized endocytic pathway in the cell. This internalization process involves the coat protein clathrin, the adaptor protein AP2 and a number of accessory proteins. All endocytic events involve a fission step, when the membrane is severed. Fission during clathrin-mediated endocytosis is regulated by the large GTPase dynamin. Dynamin self-assembles into spirals on lipid membranes and is believed to functions as a mechanochemical enzyme, severing the membrane by force generated by a GTP hydrolysis-dependent conformational change in the spiral structure. Clathrin-mediated endocytosis is the main pathway to recycle synaptic vesicles after exocytosis in the nerve terminal. Synaptic vesicle recycling is essential for sustained neurotransmission. It occurs in the periactive zone of nerve terminals. Several endocytic proteins are known to migrate from the synaptic vesicle cluster, where they reside at rest, to the periactive zone upon stimulation and Ca2+ entry. Dynamin is dependent on interactions with SH3-domain containing proteins to localize to sites of endocytis in non-neuronal cells. In the nerve terminal, dynamin interacts with a number of SH3-domain containing proteins, but the exact roles of these interactions are not known.

In this thesis, we investigate how the dynamin-binding proteins intersectin, endophilin and syndapin regulate the recruitment of dynamin to the periactive zone and the function of dynamin during fission to recycle synaptic vesicles.

We show that dynamin co-localizes with intersectin and endophilin in the synaptic vesicle cluster at rest and at clathrin-coated pits in the periactive zone in stimulated nerve terminals. Intersectin is important to regulate the amount of dynamin that is recruited to the periactive zone and to scaffold the endocytic process via the interaction with the alpha- and beta-appendages of the clathrin adaptor protein AP2. The AP2-intersectin interaction regulates the ability of intersectin to bind synaptojanin, which promotes uncoating of synaptic vesicles after they have been severed from the plasma membrane.

Intersectin and endophilin are required for proper dynamin organization at necks of ccps during clathrin-mediated endocytosis. Endophilin and dynamin assemble into a complex on membrane necks, which is required for fission.

Intra-molecular interaction between the SH3- and the F-BAR-domain of syndapin regulates its membrane tubulation activity in vivo and in vitro. This autoinhibition is disrupted by interaction with dynamin.

We propose that intersectin, endophilin and syndapin participate in the synaptic vesicle recycling process by regulating the amount of dynamin that is recruited to the periactive zone, by targeting dynamin to necks of ccps and by mediating the assembly of a pre-fission complex, which regulates membrane scission.