Morphofunctional changes at the active zone during synaptic vesicle exocytosis

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

Automated summary

This study used cryo-electron tomography to observe the intermediate steps following synaptic stimulation, and found that during the early fusion stage after stimulation, synaptic vesicles undergo morphological changes to establish contact with the plasma membrane. The subsequent late fusion stage results in fusion pore opening and collapse of the synaptic vesicles. These morphological observations likely correspond to the transition of synaptic vesicles from one functional pool to another.