Mechanistic Insights into the Role of Munc13 in Synaptic Vesicle Docking, Priming, and Fusion

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2019-03-06

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Quade, Bradley Jackson

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Abstract

Neurotransmitter release is a fundamental aspect of neuronal communication that relies on the fusion of synaptic vesicles with the presynaptic membrane. These fusion events are tightly regulated by the influx of Ca2+, which is sensed by the complex protein machinery at the axon terminal. In order for these Ca2+-mediated fusion events to occur in the correct time and place, protein machines interact with neurotransmitter filled vesicles to dock and prime them for release. Munc13 is one of the essential components of the docking, priming, and fusion machinery. To understand the role of Munc13 in docking and priming I attempted to structurally characterize the MUN domain and SNARE protein interactions using nuclear magnetic resonance spectroscopy. Paramagnetic relaxation enhancement and pseudocontact shift experiments were performed to identify the binding site of SNAREs or the SNARE complex on the MUN domain and in both cases the data suggested that there may be binding in multiple locations or that the interactions are promiscuous. I also attempted to crystallize the C2C domain of Munc13 alone and in the context of larger fragments. I was able to grow crystals of various fragments of Munc13 containing C2C and adjacent domains, but these crystals were fragile and diffracted poorly. In lieu of a crystal structure, I modeled the C2C domain based on homologous C2 domains and performed sequence conservation analysis to identify functionally important regions of C2C that may bind membranes. Using structural information coupled with reconstitutions, dynamic light scattering, and cryo-electron tomography I explored the functional relevance of membrane binding within the C1, C2B, and C2C domains of Munc13. The C2C domain was identified as a critical component of Munc13 that enables bridging between liposomes with synaptic vesicle and plasma membrane composition in vitro and this bridging ability is integral for synaptic vesicle docking in vivo. The C1C2B area has a large membrane binding interface that changes depending on the ligands present in the system and this enables Munc13 to modulate the distance between membranes. The ability of Munc13 to regulate the distance between membranes in response to ligands may underlie its role in synaptic plasticity.

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