3D MINFLUX combined with DNA-PAINT resolves the arrangement of Bassoon at active zones

Neurotransmitter release and membrane retrieval at active zones require precise spatial and temporal coordination relying on an intricate molecular machinery. However, the exact nano-structural organization of this machinery is not yet fully elucidated. Here, we used 3D MINFLUX combined with both spectral demixing and DNA-PAINT to analyze the positioning of the scaffolding protein Bassoon at presynaptic active zones of glutamatergic spine synapses of hippocampal neurons, achieving a localization precision of 5 nm in 3D. This approach allowed us to visualize directly the distribution of N-terminal and the C-terminal regions of Bassoon, and demonstrates that Bassoon exhibits an orientation at the active zone, where the C-terminal region is directed toward the synaptic cleft and the N-terminal region towards synaptic vesicles. Having demonstrated this spatial configuration in an exemplary fashion for endogenous and recombinant Bassoon molecules, our study paves the way towards molecular-scale resolution analysis of other prominent proteins of the presynaptic release machinery.