Orientation of full-length single-tagged constructs at TGN38

To verify whether the introduction of fluorescent tags on both the termini of all transfected Bassoon molecules creates an artificially exaggerated orientation of the molecule at TGN, single-tagged Bassoon constructs were transfected and imaged, following the standard protocol used for Figures 10 and Figure 16.

In addition to rule out the possibility of varying avidities of the RFP and GFP nanobodies influencing the orientation results obtained from the mRFP-mEGFP transfections (Figure 17), RFP-tagged full-length mRFP-Bsn and Bsn-mRFP constructs were compared.

The N-termini of Bassoon visualized by the RFP tag of mRFP-Bsn is observed as white, punctate, colocalized signals that have a significantly higher population of signals colocalizing to the TGN38 (84.8% ± 4.6% SD), in contrast to the large population of non-colocalizing Bsn-mRFP signals present. Although 44.4% ± 2.4% SD population of the all Bsn-mRFP signals were seen to colocalize with TGN38 signals and were distributed within 0—100nm distance category at an average distance of 50.3nm ± 3nm from the TGN lamella. These signals represented only a 0.45 faction ± 0.03 SD of the total signals/image. The majority of the Bsn-mRFP signals, were in close proximity but not at the TGN38, was distributed within the 101nm—1𝜇m distance category at an average distance of 192nm ±11.6nm from the TGN38 and represented the 0.55 fraction (±0.03 SD) of total population of its signals. The inverse observation is apparent for the highly colocalized mRFP-Bsn signals that are predominantly distributed within the 0—

100nm distance category at an average distance of 30.5nm± 6.9nm from the TGN lamella, representing the 0.84 fraction ± 0.04 SD of the total signal population. While the significantly lower fraction of mRFP-Bsn signals are distributed within 101nm—1𝜇m distance category at an average distance of 154nm ± 12nm from the TGN, representing the population fraction size of 0.15 ± 0.04 SD (Figure 18 and Appendix G).

These results are similar to those obtained from the double-tagged Bassoon constructs and show that despite the number and type of tag used the N-termini of Bassoon molecules are largely colocalized and oriented towards the TGN38 lamella while C-termini of Bassoon are mainly localized close to the TGN38 and appear to be oriented away from the TGN38 lamella.

Results Orientation of full-length Bassoon constructs to the TGN

Figure 18: Orientation of mRFP tagged full-length Bassoon constructs at the trans-Golgi network (TGN).Transfected DIV7 hippocampal neurons immunostained for the either termini of full-length Bassoon with RFP-nanobody-Atto594 and the TGN38 marker. Two-color deconvolved 10μm X 10μm STED images of the N–terminus of Bassoon (A) and the blow-ups of its inset (B—D) or the C–terminus of Bassoon (A) and the blow-ups of its inset (F—H). I represents the colocalization quantification, and J represents the distribution quantification at and away from TGN38 lamella i.e. 0—100nm or 101nm—1μm, respectively. Data are represented as mean ± SD, N=10 cells from two separate experiment, statistically tested with a one-way annova with the Tukey’s multiple comparison’s post-hoc test *p < 0.05 and ***p ≤ 0.001. Scale bars 1μm (A—H).

3.5.3. Orientation of full-length single and double-tagged constructs at Syn6

To ascertain whether the orientation of Bassoon is specifically regulated to the TGN38 domain or whether the orientation of Bassoon is uniform all over the TGN lamella, the orientation of double-tagged and single-tagged full-length Bassoon constructs were visualized at another TGN lamella protein; Syntaxin6 (Syn6), Syn6 much like TGN38 is a protein present at the lamellae of the trans-Golgi network that is involved in sorting and trafficking of proteins from the TGN to the plasma membrane and back. Syn6 is a 255 a.a.-long protein that possesses a N-terminal soluble SNARE domain that localizes Syn6 to the TGN, and possess an additional sorting motif that promotes retrograde transport of the Syn6 positive vesicles. It also possesses a hydrophobic C-terminal transmembrane region that acts as a membrane anchor and regulates the sorting of proteins at the TGN lamella^107,108. The antibody toward Syn6 targets an epitope spanning 6—136 amino acids that contains its N-terminal TGN-targeting motif and therefore works as an additional marker to label the TGN lamella. Images of the single- and double-tagged Bassoon constructs were imaged and processed with the standard STED imaging protocol.

Results Orientation of full-length Bassoon constructs to the TGN Remarkably, the N-terminally tagged Bassoon signals of both the mRFP-Bsn-mEGFP and mRFP-Bsn construct have high colocalizations with the Syn6 signals at 71.8% and 75.8% respectively, while the C-termini of the mRFP-Bsn-mEGFP (23.8%) and Bsn-mRFP (43.7%) construct have significantly lower number of colocalized signals.

These results reflect the same colocalization patterns as those observed in the TGN38 images and therefore expectedly present with similar signal distribution patterns.

Wherein, significantly higher fraction of signals were distributed within 0—100nm distance category for the N-termini of single- (0.8 population fraction ± 0.03 SD) and double-tagged Bassoon molecules (0.71 population fraction ± 0.02SD), while the major fraction of C-terminal Bassoon signals from the single- (0.62 population fraction ± 0.03 SD) and double-tagged (0.65 population fraction ± 0.04 SD) images lay in the 101nm—1𝜇m category (Figure 19).

This result reflects that Bassoon molecules are orientated robustly all over the TGN lamella with their N-termini localizing within 0—100nm of the TGN lamella, irrespective of the application of either TGN38 or Sny6 marker, and are present as highly colocalized signals even after being resolved by STED. The C-termini of tagged Bassoon molecules are also always seen localized close to the TGN lamella, irrespective of the marker used, and are present as clearly resolved signals that are majorly distributed within the 101nm-1𝜇m distance category.

Results Orientation of full-length Bassoon constructs to the TGN

Figure 19: Orientation of single and double-tagged full-length Bassoon constructs at the trans-Golgi network marker: Syntaxin 6 (Syn6).

Transfected DIV7 hippocampal neurons immunostained for the either or both termini of full-length Bassoon with RFP-nanobody-Atto594 and/or GFP-nanobody-Atto647 with the Syn6 marker. Two-color, deconvolved, 10𝜇m X 10𝜇m STED images and their insets, representing the N–terminus of double-tagged and single-tagged Bassoon constructs in panels A—D and I—L, respectively.

Similar STED images of the C–terminus of the double-tagged and the single-tagged Bassoon constructs, are available in panels E—H and M—P, respectively. Graph Q quantifies the amount the colocalization and graph R quantifies the signal distributions at and away from TGN38 lamella i.e. 0—100nm or 101nm—1𝜇m, respectively. Data are represented as mean ± SD, N=10 cells from two separate experiment, statistically tested with a one-way annova with the Tukey’s multiple comparison’s post-hoc test **p ≤ 0.01 and ***p ≤ 0.001. Scale bars 1𝜇m (A—P).

Results Orientation of full-length Bassoon constructs to the TGN

3.5.4. Detailed orientation of Bassoon molecule at the