Synaptic vesicle-bound molecular bridges organize sequential vesicle states along parallel pathways

Multiple complexes and biochemical pathways are involved in priming synaptic vesicles and subsequent neurotransmitter release. While atomic models of reconstituted complexes, interaction data and physiological experiments were essential for our understanding of the mechanisms underlying synaptic transmission, they lack the combination of the native preservation, comprehensive view and a high level of detail to resolve how the actions of individual complexes integrate to give rise to cellular function. We employed cryo-electron tomography to simultaneously image multiple presynaptic complexes at a single nanometer resolution in their native composition, conformation and environment, and to determine their spatial organization. Our results show that Munc13- and SNAP25-dependent tethers localize vesicles with a single nanometer precision, thus defining sequential vesicle states that immediately precede neurotransmitter release. The transition between the states involves two parallel biochemical pathways and is associated with a conversion to SNARE complex-dependent tethers and a loss of protein connectors that interlink vesicles. These results provide an example of a cellular function performed by a large molecular assembly comprising multiple, spatially separated complexes and provides a framework for further exploration of such assemblies. ### Competing Interest Statement The authors have declared no competing interest.