The role of multifunctional scaffolding proteins in the synaptic vesicle cycle

Fast synaptic transmission occurs at specialized junctions between neurons referred to as chemical synapses. Action potentials induce an influx of calcium ions into presynaptic terminals, which contain neurotransmitter-filled synaptic vesicles (SVs), triggering fusion of the vesicles with the plasma membrane and resulting in the release of neurotransmitters. After fusion SVs have to be recycled and refilled to maintain neurotransmission for a certain period of time. Clathrin-mediated endocytosis serves as a major mechanism for synaptic vesicle recycling. It occurs at the periactive zone and relies on a set of proteins such as clathrin and clathrin adaptors, which are essential for clathrin coat assembly, and the GTPase dynamin, which is required for budding of the newly formed vesicles from the plasma membrane. Multiple accessory and scaffolding proteins coordinate the assembly of the clathrin vesicles. In this thesis, the functional role of the scaffolding proteins Dap160 and Eps15 in the synaptic vesicle cycle was investigated. The genetically tractable Drosophila neuromuscular junction (NMJ) was used as an experimental model. Several new methodological approaches, such as high pressure freezing, freeze substitution, and a correlative immunogold technique were developed or adapted in this work to study the Drosophila synapse. These approaches allowed for the characterization of the structure of the synapse and the organization of vesicles, and for the first time provided 3-dimensional reconstruction of the presynaptic specialization. The subcellular localization of Dap160 and Eps15 was determined. Biochemical experiments revealed that they form a molecular complex. Structural and functional analysis of Drosophila dap160 and eps15 mutants showed that these proteins have a dynamic localization in the nerve terminal: both molecules reside in distal pool of SVs at rest and relocate to the periactive zone during synaptic activity. dap160 and eps15 single and double mutants display defects in synaptic vesicle recycling. Physiological experiments show that both proteins are required to maintain synaptic transmission at high activity rates. Genetic disruption of the interaction between the Dap160-Eps15 complex and the GTPase dynamin results in abnormal distribution of dynamin immunoreactivity at the periactive zone during stimulation. We conclude that the Dap160-Eps15 molecular complex is essential to concentrate dynamin at the periactive zone during synaptic activity. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to in the text according to their Roman numerals: I. Koh, T. W. , Korolchuk, V. I. , Wairkar, Y. P., Jiao, W. , Evergren, E. , Pan, H. , Zhou, Y. , Venken, K. J., Shupliakov, O. , Robinson, I. M. , O'Kane, C. J., and Bellen, H. J. (2007) Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development, J Cell Biol 178, 309‐322. II. Jiao, W. , Shupliakov, A. , and Shupliakov, O. (2010) A semi‐correlative technique for the subcellular localization of proteins in Drosophila synapses, J Neurosci Methods 185, 273‐279. III. Jiao, W. , Masich, S. , Franzen, O. , and Shupliakov, O. (2010) Two pools of vesicles associated with the presynaptic cytosolic projection in Drosophila neuromuscular junctions, J Struct Biol 172, 389‐394. IV. Jiao, W. , Winther. Å. , Koh, T. W. , Vorotsova, O. , Bellen, H. J., Shupliakov, O. (2011) Dynamin targeting to the periactive zone in Drosophila synapses is mediated by Dap160, Resubmitted to J Cell Biol.