Thursday, March 13, 2025
3/13/2025
Synaptic transmission between pre- and postsynaptic neurons occurs when the presynaptic neuron terminal is temporarily depolarized upon an action potential, opening Ca2+ channels near the active zones of synapses. Because the extracellular Ca2+ concentration is much higher than the cytoplasmic concentration, Ca2+ flows into the cytoplasm, triggering fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane in less than a millisecond. Upon fusion, neurotransmitter molecules are released into the synaptic cleft, and then bind to receptors located in the postsynaptic membrane. Finally, the fusion machinery is recycled for further rounds of fusion in the presynaptic cell. Major questions about the molecular mechanisms of membrane fusion and protein recycling remain. The architecture of the fusion machinery between the synaptic vesicles and plasma membranes is unknown, and the molecular steps after Ca2+-triggering are unknown. Furthermore, our understanding of the molecular mechanisms governing synaptic release probability and presynaptic plasticity is incomplete. Obtaining three-dimensional images of synaptic proteins within their natural membrane environment will be an essential step towards answering these questions. We propose a stepwise, bottom-up approach starting with simpler systems and moving to increasingly more complex systems. First, we will employ a hybrid (ex vivo / in vitro) approach where synaptic vesicles are isolated from mouse brain homogenates and combined with synthetic acceptor vesicles. Functional tests of this hybrid system will be performed using a new single vesicle fusion assay that discriminates between different stages of membrane fusion and includes many presynaptic proteins, including but not limited to SNAREs, synaptotagmin, and complexin. The contact sites between isolated synaptic vesicles and synthetic vesicles will be imaged by cryo-electron tomography (cryo-ET) followed by subtomogram averaging to reveal the architecture of these presynaptic complexes in their membrane environment. Next, we plan to image the equivalent membrane contact sites in both isolated synaptosomes and in synapses of neuronal cultures grown on EM grids. We anticipate that reconstructions of presynaptic complexes obtained with the hybrid approach are the starting point for locating such complexes in synaptosomes and in synapses in neuronal cultures. These in vivo reconstructions might reveal new molecular interactions or associations. After fusion, the AAA+ protein NSF and associated SNAP co-factors are essential for disassembly of SNARE complexes and for quality control of newly formed SNARE complexes (in conjunction with Munc18 and Munc13). Previously, we determined single-particle cryo-electron microscopy (cryo-EM) structures of the complex of NSF, αSNAP, and the neuronal SNARE complex under non-hydrolyzing conditions. We now aim to investigate the molecular details of disassembly, what conformational changes are involved, and how ATP hydrolysis is coupled to these conformational changes by cryo-EM.
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