|dc.description.abstracteng||Neurotransmitter release sites at the presynaptic terminus membrane, known as the active zones (AZs), are surrounded by synaptic vesicle pools and a dense network of five cytomatrix of the active zone (CAZ) proteins. At CNS synapses, Munc13s, RIMs, CAST/ELKS, Bassoon, and Piccolo predominantly form the CAZ scaffold, and together have been shown to promote short- and long-term plasticity by binding to Ca2+ channels and enabling priming and docking of synaptic vesicles to the presynaptic membrane. Even though the components of the CAZ are known, how they are exactly assembled opposite postsynaptic specializations is not yet understood.
It has been shown that AZ proteins (AZPs) are transported on 80nm dense-core vesicles called Piccolo/Bassoon transport vesicles (PTVs), to synapses in aggregates together with synaptic vesicles. In addition, Golgi-derived AZ precursor vesicles that transport these proteins have been reported to take different paths out of the Golgi and carry only a small subset of AZPs, although how all AZPs reach and generate a complete and functional AZ at the presynaptic terminus is still under investigation. These observations suggest traffic of a range of different transport vesicles, carrying subsets of AZPs to synaptic sites, and indicates that the mechanisms influencing the final assembly of AZPs at the presynaptic terminus may be predetermined as early as their sorting and loading onto transport vesicles at the Golgi.
To address this hypothesis, this study, examines the localization of endogenous AZPs, using super-resolution microscopy, for the first time at Golgi substructures, the soma, and in the developing axons of hippocampal neurons. AZPs are specifically localized at and around their respective Golgi lamella, in a range of signal sizes that correspond to different loaded transport-carrier types, and present low co-localizations, with one another, in developing axons. This distribution signifies the importance of early sorting and loading of preassembled AZP subsets in the soma. In order to understand the underlying mechanisms that dictate the specific localization of CAZ proteins, a detailed study of the nanostructural orientation and organization of AZPs, at different cellular locations, is required, but hampered by the limitations imposed by the use of primary and secondary antibodies. To overcome this technical limitation, I introduce, characterize, and use new full-length second-generation Bassoon constructs that are optimized, with respect to their targeting behavior in neuronal cells, and are endowed with tags that can be detected with very small camelid antibodies, so called nanobodies, for super-resolution imaging.
Bassoon is one of the largest CAZ proteins and among the first AZPs to be incorporated at young synaptic sites. It is known to bind to other AZ proteins
in the CAZ scaffold, and provides structural stability to the CAZ scaffold by downregulating local ubiquitination. This suggests a central role of Bassoon in CAZ scaffold generation. In addition, Bassoon is also the mammalian AZP with the largest cohort of mutant and full-length constructs available, making it an ideal candidate for this study.
STED and FLIM imaging show that full-length Bassoon molecules possess an open and extended conformation at the TGN and are organized 6—20nm from the TGN with neighboring N-termini of molecules in close proximity to each other. Further, these studies show that the first 94 amino acids of Bassoon’s N-terminus, but not its myristoylation motif, determines its correct subcellular localization to the TGN, while Bassoon’s CC2 domain is sufficient for recruiting the protein to the Golgi. A novel conformation change is observed as the Bassoon molecule travels from the Golgi to synaptic sites, where the molecule appears to lose its extended conformation during trafficking on ChromograninA-positive PTVs, and returns to its extended orientation at synaptic sites. Within these sites, in CAZ scaffold, Bassoon molecules have been previously shown to be oriented with their N-termini extending 80nm into the presynaptic bouton and their C-termini positioned around 50nm from the presynaptic plasma membrane. In this study I show that the N-termini of neighboring Bassoon molecules are organized in close proximities of ≥5nm from each other. This result suggests that the organization of Bassoon molecules within the CAZ scaffold closely resembles triangular dense projections regularly observed in EM images of CNS presynaptic sites. Therefore the orientation and the organization of Bassoon molecules promotes structural stability by inhibiting localized ubiquitination and forms the backbone that other AZPs bind to within the CAZ scaffold.
In conclusion, the data reported here suggest that the orientation and organization of Bassoon molecules plays an important role in promoting local subcellular mechanisms from influencing its localization and sorting at the TGN to providing structural stability to the CAZ scaffold, while its change in conformation on its journey, to the CAZ, highlights the first step in understanding the sequence of mechanisms involved in mammalian AZ assembly and synapse maturation.||de