4.1.1 Active zone proteins selectively localize to different Golgi subcompartments
AZPs are first observed in young neurons localized to the Golgi and selectively get recruited to specific Golgi subcompartments. Bassoon, Piccolo and ELKS2 have been shown to specifically localize to the trans-Golgi Network, Munc13-1 is
Discussion Recruitment and sorting of AZPs at the TGN observed at the cis-Golgi lamella, and RIM1𝛼 is diffused in the soma and in young neurites (Figure 3, Figure 4 and ref.32). While Rim1𝛼’s localization is only observed later in development, on RIM-Neurexin-CASK-voltage-gated Ca2 channels (VGCCs) transport units in the axon3, the other CAZ proteins undergo sorting and assembly onto transport carriers at their respective Golgi compartments. Using STED microscopy, I observed endogenous AZPs at their designated Golgi compartment as punctate signals decorating regions at and around the Golgi lamella, and, unlike previous observations with confocal microscopy, these AZP localizations were not uniformly spread all over the Golgi signals. This localization of AZPs suggests the presence of specialized sites at their Golgi subcompartment where the proteins are sorted and loaded onto unique transport carriers. The properties of the transport carriers, conferred by the Golgi compartment they are generated from, will ultimately define the subsets of AZPs that co-traffic out together from the soma and the route of transport they take down the axon based on the microtubule adaptor proteins they possess (Figure 28).
Similar localizations of invertebrate AZ proteins Liprin and Syd2111 and presynaptic proteins such as synaptic vesicle marker 2 (SV2)112, synaptophysin113, SNAP25, and VAMP2114 have also been reported at the TGN network with light and confocal microscopy. In addition, TGN-derived vesicles have been shown to supply developing inhibitory synapses by shuttling GABAA
receptors, their associated cytoskeletal linker protein Gephyrin, and ATPase N-ethylmaliemide-sensitive factor (NSF) (that activates SNARE proteins) to the synaptic sites115.
These and many more studies add to the evidence that most AZ proteins and synaptic proteins originate from the TGN96,113,115116–118, although no known link between cis-Golgi traffic and supply to the presynaptic terminus has been documented. Nevertheless, proteins undergoing a first round of sorting at the cis-Golgi are known to be either internally trafficked, through all the cis-Golgi lamellae, to the TGN for a second round of sorting or a subset of certain proteins are also known to be transported back to the ER after only the first round of sorting119. Interestingly, glutamate receptor NMDAR subtypes and their associated postsynaptic adaptor proteins CASK and SAP97 have been shown to be transported from the cis-Golgi and localized at the ER. From the ER these proteins take an unconventional secretory pathway to the plasma membrane where the proteins first get trafficked to axonal and dendritic Golgi outposts, which eventually traffic these proteins to presynaptic and post-synaptic sites120. As Munc13-1 is the first documented AZP to be localized at the cis-Golgi and does not colocalize to the TGN, it might take a similarly unconventional route, like the NMDAR receptors, out of the soma to the presynapse (Figure 28). This choice of unconventional localization may hint at a mechanism that brings a
Discussion Recruitment and sorting of AZPs at the TGN greater diversity of proteins to the presynapse, temporally recruits subsets of AZPs for each step of the CAZ assembly, and fosters heterogeneity within pre-assembled AZP subsets co-trafficking on small vesicle clusters.
Figure 28: A diagrammatic representation of AZPs localizations to different Golgi subcompartments. This diagram depicts a schematic of transport precursors being generated at the cis-Golgi vesicles carry Munc13-1 and TGN vesicles carrying subsets of ELKS2, Piccolo and Bassoon which may come in close proximity in the soma for co-transport down the axon.
4.1.1 The N-terminus of Bassoon localizes it to the TGN
The localization of Bassoon, Piccolo and ELKS to the TGN may be conferred by a direct interaction site on the protein or via an adaptor protein that is localized at the TGN lamella. N-terminally tagged RFP-Bsn specifically localizes to the TGN marker TGN38 but not to the trans-Golgi lamella marker 𝛽-1,4-galctosyltransferase 1, positioning full-length Bassoon to the sorting TGN compartment (Figure 16). The C-terminus of full-length Bassoon does not localize close to the TGN lamella but also does not localize at the trans-Golgi lamella (Appendix F). Therefore Bassoon molecules are not localized between the trans-Golgi lamella and the TGN but are rather localized on a more distal trans-Golgi lamella.
The TGN network constitutes the last three cisternae of the Golgi and has subdomains that are characteristic for different resident proteins. TGN cisternae 5
Discussion Recruitment and sorting of AZPs at the TGN and 6, of the whole Golgi stack, are asialoglycoprotein positive and are decorated with 60nm clear-core vesicles with lace-like-coats that are regulated by protein kinase D activity. These vesicles are believed to transport cargoes en route to the plasma membrane. On the other hand, mannose-6-phosphate protein positive cisternae 7 is known as the clathrin-coat subdomain of the TGN, and produces
~100 nm clathrin-coat buds that are regulated by ADP-ribosylation facto family of GTPases ARFs) and GGA (Golgi-localized, gamma-ear containing, ADP-ribosylation factor binding) proteins121,122.
Since neither full-length Bassoon termini are localized close to the trans-Golgi lamella (Figure 16, Figure 17—Figure 19, and Appendix F) and endogenous Bassoon majorly presents itself in signal sizes of 95-150nm (Figure 5), it seems likely that Bassoon molecules may be localized to the furthest TGN subdomain of cisternae 7. In addition, Bassoon transported on a range of different DCVs subtypes such as CGA-positive vesicles, PTVs and Syt4-positive vesicles (Figures 6, 25 and ref 69), which may also possess clathrin coats, since there is strong evidence from electron micrographs of neuroendocrine cells demonstrating budding DCVs possessing clathrin coats123,124. It therefore would be interesting to see if Bassoon carrying PTV transporters are clathrin-coated and whether their fission from the TGN lamella is ARF- or GGA-protein mediated.
Full-length Bassoon is highly colocalized to TGN markers through its N-terminus and not though its C-terminus (Figure 16—Figure 19, and Appendix F), making the N-terminus of Bassoon its localization and possibly interaction domain for sorting at the TGN. The myristoyl motif, at the second amino-acid position of the N-terminus, was found to not influence its localization at the TGN though another site within the N-terminal domain might (Figure 23). This would require a systematic characterization of an array of N-terminal deletion constructs to pinpoint the exact site that influences Bassoon’s localization, although it might very well be possible that the N-terminus of Bassoon never integrates into the TGN membrane but rather is attached to the TGN via an adaptor protein or is directly loaded onto constitutively formed nascent buds from the TGN lamella.