4.4.1 Current concept: Bassoon possesses an extended conformation at the presynaptic terminus
Bassoon (420kDa) and Piccolo (550kDa) are the two largest proteins in the CAZ, and both molecules have been shown to possess an extended orientation at matured presynaptic sites in mammalian neurons76,77. In-silico modeling predicts that both molecules contain a large number of proline and glycine residues, which prevent folding of these proteins as a whole, but also contain interspersed compact regions that hold the Zn-finger, coil-coil, and Piccolo-Bassoon homology domains, as a result of which their entire molecule elongates to about 80 nm (Figure 29,B and ref24).
Discussion Orientation of the Bassoon molecule
Figure 29: Structure and orientation of Bassoon and Piccolo proteins at the presynaptic terminus.
A and B, In-silicio model of Bassoon and Piccolo proteins with interaction sites and binding partners (Modified from Gundelfinger et al, 2015). C and D, Gold labeled EM images of N- and C- termini of Piccolo protein, on the apical and base locations of the dense projections (DPs) in mammalian presynaptic sites (Modified from Limbach et al, 2011). E and F, represent triple colour-3D STORM images of Bassoon and Piccolo at side view synapses (Modified from Dani et al, 2010). G is a representation of CAZ and pre- and postsynaptic protein distances from the synaptic cleft. (Modified from Dani et al, 2010 and enhanced with information on Munc13-1, RIM1 and Ca2+ channel distances from Limbach et al, 2011).
In a recent study, triple-color 3D STORM images of cortical synapses labeled using a Bassoon monoclonal antibody spanning the region between 765—
1001a.a. (closest antibody to the N-terminus of Bassoon), a C-terminal polyclonal Bassoon antibody spanning the last 338 a.a. of the molecule, and a Homer1 antibody (to define postsynaptic membrane and the synaptic cleft size) were used to determine Bassoon’s orientation at the synapse (Figure 29E,F). These images showed that the N-terminus of Bassoon is localized at ~78nm, and ~30nm further away from the synaptic cleft than the C-terminus of Bassoon signals, which is localized ~43 nm from the synaptic cleft. Bassoon and Piccolo signals were shown to spatially occupy similar locations in the CAZ as Piccolo’s N- and C-terminal regions were located roughly 86nm and 44.4nm, respectively, from the synaptic cleft76.
The orientations of Bassoon and Piccolo molecules recorded by STORM imaging also fit with results from another study that studied the localization of many immunogold electron micrographs of cerebellar rat neurons labeled for a range of antibodies generated against short sequences within Aczonin/Piccolo molecule and for Bassoon’s C-terminus. 70% of the total N-terminal sequences of Piccolo were observed localized within 73—79nm from the plasma membrane (PM),
Discussion Orientation of the Bassoon molecule while the 70% of the C-terminal Piccolo sequences lay within 33—39nm, and Bassoon’s C-terminal antibody signals were observed positioned within 35—
37nm from the PM (Figure 29C,D and ref77).
Both these studies, using two different super-resolution imaging techniques, have shown that the N-termini of Bassoon and Piccolo molecules within an AZ complex are at least 80nm from the PM, their C-termini face the PM, and are roughly 30—40nm from the presynaptic membrane.
Ultrastructural imaging of the same synapses also revealed AZPs that bind to C-termini of Bassoon and Piccolo, in particular the N-C-termini of Munc13-1 and RIM1𝛼, localized 19nm from the PM and the cytoplasmic loop of the P/Q type Ca2+ channels were localized 20nm from the PM, positioning the entire AZ complex within a minimum distance range of 20—80nm from the PM (Figure 28G and ref77). These studies revealed a range of additional characteristics that define the CAZ ultrastructure such as the following: the presence of 60nm high and 30nm wide dense projections (DPs) at the presynapses of chemically fixed EM images that reflect the dimensions of a collapsed CAZ filament structure (Figure 28C/D and ref77), the distances of the C-termini of Piccolo and Bassoon noted are similar to the diameter of synaptic vesicles (SVs) (i.e. ∼35–40 nm)3,77, and that none of the AZPs in the CAZ have transmembrane regions that warrant their presence at the PM40,70. This shows that the CAZ structure localizes within the presynaptic bouton but floats at a minimum 20—30nm distance, reflective of the size of a SVs docked, on top of the PM.
This extended orientation of Bassoon and Piccolo molecules seem to inherently influence the function of the CAZ, and are also seen in invertebrate synapses.
Bruchpilot, a Drosophila CAZ core component that is evolutionarily unrelated to Bassoon and Piccolo, has also been shown to possess an extended orientation at the neuromuscular junction. Although the orientation of Bruchpilot differs from Bassoon and Piccolo’s, as its N-terminus is closer to the presynaptic membrane, it vertically extends 40—65nm into the presynaptic bouton and matches Bassoon and Piccolo heights in mammalian presynaptic membrane77,137. This shows that CAZ structure needs to be accessible to resting and recycling SV pools in the presynaptic terminus, and Bassoon and Piccolo, like Bruchpilot at invertebrate synapses, provide the structural backbone of the CAZ scaffold with their extended conformations.
4.4.2 Bassoon also possesses an extended conformation at the TGN but it is inversely oriented
In this study, Bassoon was visualized at its first station the TGN and was revealed to possess an extended conformation at this structure. This is a novel observation as it ties the orientation of Bassoon at it its initial and final station in its journey, shows that Bassoon possess a similar conformation at both sites and
Discussion Orientation of the Bassoon molecule 229.4nm ± 5.3nm SD from the closest TGN38 marker. Meanwhile, 63% of the tagged C-termini signals of the same full-length construct were localized on average around 210.6nm ± 13nm SD, and the remaining 37% of C-terminal tag signals were localized around 60.4nm ± 6.8nm SD from the TGN38 marker. This predicts that at and around the TGN, there is a mixed population of Bassoon molecules that are being oriented at the TGN and those that are already loaded onto transport carriers. The closest signals of full-length Bassoon’s N-terminal tag were seen localized ∼30—40nm from the TGN and the closest C-terminal signals were seen around 60—67nm from the TGN membrane. These represent the molecules that are being oriented at the TGN and show that the N- and C-termini of Bassoon are positioned within 30—40nm of each other (Figure 17). These results remarkably concur with the 30—40nm extension of the Bassoon molecule seen in mammalian presynaptic boutons, from their EM and STORM orientation studies76,77. Bassoon’s equidistant localization on top of CGA-positive DCVs, seen further down the axon (Figure 25 and Figure 17).
These localizations of full-length Bassoon are extremely reproducible and robust since similar distances for single-tagged RFP-Bsn and Bsn-RFP constructs were also observed, along with a similar distribution of all full-length Bassoon constructs to a different TGN marker: Syn6 (Figures 18 and 19). The subset of single-tagged Bassoon signals being orientated at the TGN were localized at 30.5nm± 6.9nm SD (for 85% of the RFP-Bsn) and at 50.3nm ± 3nm SD (for 45%
of the Bsn-RFP) from the closest TGN marker, while the fraction of signals on transport precursors were localized at 154.1nm ± 12.06 nm SD (for 15% of RFP-Bsn) and 192nm ±11.6nm SD (for 55% of the Bsn-RFP) (Figure 18).
Overall these results show that Bassoon orients itself with its N-terminus at the TGN membrane while its C-terminus is at least 30—40nm form the TGN lamella.
Although the extension of the Bassoon molecule is remarkably similar to its reported extension within the CAZ, the orientation of the molecule is inversed between the two stations. As the Bassoon molecule does not possess transmembrane regions that may integrate the molecule at either the TGN lipid
Discussion Orientation of the Bassoon molecule bilayer or the PM, and its N-terminal myristoyl motif is not involved with its localization to the TGN membrane, one may assume that Bassoon’s orientation is not influenced by a docking site within either membrane at the two stations.
The inverse orientation is therefore a reflection of the roles and the local subcellular mechanisms the Bassoon molecule is involved in, at both stations.
At the TGN, the extended orientation of Bassoon may promote sorting mechanisms and aid in exposing Bassoon’s central CC2 domain. The CC2 domain has been shown to sufficiently recruit Bassoon molecules to the Golgi (Figure 22), and, while at the TGN, promotes oligomerization of Bassoon-Bassoon and Bassoon-Bassoon-Piccolo clusters that get loaded onto a range of transport carriers thereby promoting subsets of preassembled AZP to be co-trafficked.
Additionally upstream from the CC2 domain lies the CtBP1 interaction site, which should become available for binding as Bassoon’s conformation becomes extended at the TGN membrane thereby promoting CtBP1 mediated sorting. It therefore would be interesting to visualize the average localization that CtBP1 occupies at the TGN using full-length Bassoon as a ruler.
4.4.3. The limitations of different super-resolution microscopy techniques in understanding Bassoon’s orientation
Until recently, all ultrastructural imaging in the brain was done using transmission electron microscopy (TEM). The constant challenge of acquiring sufficient contrast though ultrathin sectioning, followed by the demands of serial sectioning and section alignment, have decreased the throughput of TEM sample preparation. In addition, the technique necessitates harsh sample preparations that often subject the samples to form artifacts that could obscure the true structure and localization of proteins at an ultrastructural level. The last decade has seen the rise of light microscopes created to break the resolution limit of light and resolve a much higher level of detail. STED and STORM microscopy are classical examples of such tools that have created a platform to revisit and add to existing EM data by allowing the visualization of tagged proteins at super-resolution.
In this study, I have made use of a range of full-length Bassoon constructs that have allowed me to visualize the orientation of the Bassoon molecule with great detail and accuracy. Visualizing these constructs with two-color STED microscopy (at a resolution limit of 20nm) benefits from the tags on the N- and C-termini of Bassoon being placed at the extreme poles of the protein, without hampering its localization, transport and incorporation into synapses, and allows for the use of specific nanobodies targeting the fluorescent tags, that localize the whole tag-complex within 13nm of the protein. The use of the double-tagged full-length Bassoon construct, boosted with nanobodies, and imaged with STED to visualize the localization of the N- and C-termini of Bassoon, overcomes a range
Discussion Orientation of the Bassoon molecule of disadvantages faced by the other super-resolution imaging studies performed.
One of the key advantages is eliminating the use of traditional antibodies of Bassoon for STED microscopy, which would create a 30nm cloud of signal around the protein epitope and distances the correct subcellular localization of the termini of Bassoon. STED imaging of transfected neurons also benefits from the ability to avoid the use of the harsh processing steps that are essential for processing EM images that cause the CAZ structure to collapse into DPs. When comparing my imaging paradigm with the drawbacks of STORM imaging results of Bassoon’s orientation at the presynaptic termini, it becomes clear that the latter’s results suffer from the application of two traditional antibodies targeting a region of Bassoon around its 1000th a.a. and the extreme C-terminal region and can therefore only predict, at a 30nm distance from the true localization, the distance between the 1000th and 4000th amino acids of the Bassoon molecule. It therefore would be interesting to visualize the orientation of Bassoon at mature synapses using nanobodies and the full-length Bassoon constructs and ascertain whether the first 1000 a.a. of Bassoon add to further extend the molecule into the presynaptic bouton or whether it folds to create a cap for the filament like structure that Bassoon possesses within the CAZ.
The STED imaging paradigm I use, on the other hand, is limited by the confocal resolution (~200nm) in its z-plane, in comparison 3D STORM imaging allows for a ~14nm resolution limit in the z-plane. This limitation has so far made it difficult to find in culture, pre- and postsynaptic sites in the side view conformation, which is a clear separation of pre- and postsynaptic compartments with the synaptic cleft (Figure 28E, F). This conformation of viewing synapses is important as it provides a clear marker from which to define the distance of the N- and C-termini of a molecule and can be observed with greater ease with 3D super resolution imaging. In addition, to understand the real orientation and organization of CAZ proteins in the CAZ scaffold live neurons need to imaged, for which the required dual-color RFP and GFP live STED microscopes are still being developed.