Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus

1.Bassoon is a large, 440 kDa, scaffold protein of the specialized sites mediating neurotransmitter release from presynaptic nerve terminals, called active zones. At active zones of the brain, Bassoon is arranged with its C-terminus facing the plasma membrane. In young, cultured neurons Bassoon is associated with the Golgi-apparatus, where it may assemble active zone precursors structures, but whether or not Bassoon is an extended protein at the Golgi-apparatus and whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Here, we generated recombinant Bassoon constructs for expression in neurons, with tags positioned to allow for the specific detection of N- and C-terminal regions of Bassoon by single-domain antibodies, called nanobodies. Using stimulated emission depletion (STED) nanoscopy in cultured hippocampal neurons, we confirmed that recombinant Bassoon is oriented with its C-terminus towards the active zone plasma membrane at synapses. Focusing on the soma, we found that an intramolecular tag located immediately downstream of amino acid 97 of Bassoon, showed prominent colocalization with markers of the trans Golgi network, including TGN38 and syntaxin-6. In contrast, tags located immediately downstream of the C-terminal amino acid 3938 of Bassoon showed significantly less colocalization with these Golgi-markers. The intramolecular N-terminal tag was located between 48 and 69 nm away from TGN38, while C-terminal tags were located between 100 and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon, but not its N-myristoylation sequence, were required for this arrangement. Our results indicate that at the Golgi-apparatus Bassoon is oriented with its N-terminus towards and its C-terminus away from the trans Golgi network membrane. Moreover, they suggest that Bassoon is an extended molecule at the trans Golgi network with the distance between amino acids 97 and 3938 estimated to be between 46 and 52 nm. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.