Synaptic vesicle (SV) fusion is not only tightly coordinated but also happens at a millisecond timescale. Competing models for fusion initiation and propagation suggest tight docking and hemifusion of SVs or localized lipid rearrangements leading to tip-like membrane contacts. Yet, a direct nanoscale examination of the full SV fusion sequence has been lacking. Here, we establish a workflow for timed in situ cryo-electron tomography of optogenetically stimulated mouse neurons to capture the complete SV fusion sequence – from SV recruitment to fusion pore formation, opening and collapse – with near-native structural preservation. Notably, tethered SVs directly undergo fusion initiation via stalk formation, without preceding tight docking or SV flattening. The plasma membrane forms a minimal dimple during fusion initiation, contradicting preceding models that invoke strong membrane bending prior to fusion. In addition, we observe filaments linking fusing SVs to adjacent SVs, indicating a physical link between fusion and SV resupply.
