Kravcenko, U. | Search bioRxiv

bioRxiv2025

Dynamic nanoscale architecture of synaptic vesicle fusion in mouse hippocampal neurons

During neurotransmission, presynaptic action potentials trigger synaptic vesicle fusion with the plasma membrane within milliseconds. To visualize membrane dynamics before, during, and right after vesicle fusion at central synapses under near-native conditions, we developed an experimental strategy for time-resolved in situ cryo-electron tomography with millisecond temporal resolution. We coupled optogenetic stimulation with cryofixation and confirmed the stimulation-induced release of neurotransmitters via cryo-confocal microscopy of a fluorescent glutamate sensor. Our morphometric analysis of tomograms from stimulated and control synapses allowed us to characterize five states of vesicle fusion intermediates ranging from stalk formation to the formation, opening, and collapsing of a fusion pore. Based on these measurements, we generated a coarse-grained simulation of a synaptic vesicle approaching the active zone membrane. Both, our morphofunctional and computational analyses, support a model in which calcium-triggered fusion is initiated from synaptic vesicles in close proximity to the active zone membrane, whereby neither tight docking nor an induction of membrane curvature at the active zone are favorable. Numbers of filamentous tethers closely correlated to the distance between vesicle and membrane, but not to their respective fusion readiness, indicating that the formation of multiple tethers is required for synaptic vesicle recruitment preceding fusion.

neuroscience↗

bioRxiv2024

Molecular architecture of synaptic vesicles.

Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs are formed in a clathrin-dependent manner. This process maintains the morphology and complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV1,2. However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here we used cryo-electron tomography to visualize morphological and molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram average, and showed them forming a complex with the membrane-embedded protein synaptophysin. Our bioluminescence assay revealed pairwise interactions between VAMP2 and synaptophysin and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle sizes. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with a soccer-ball symmetry. We observed a V-ATPase under clathrin cage in several isolated clathrin-coated vesicles. Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs diversity and their molecular architecture.

biophysics↗