Fig 2: Syntaxin clusters change the distribution of SNAP25.Membrane sheets are generated from PC12 cells expressing GFP-SNAP25 alone (control) or together with Stx-full/Stx-?S. Endogenous syntaxin/syntaxin constructs are recognized by an antibody raised against the molecule’s N-terminus, visualized by an AlexaFluor594-coupled secondary antibody; GFP-SNAP25 is visualized by an ATTO647-labelled GFP nanobody. (A) Representative images from the syntaxin 1A and SNAP25 channels of the control (only endogenous syntaxin), Stx-full (+ additional syntaxin), and Stx-?S (+ additional syntaxin with a shortened SNARE domain) condition. For magnified views see Figure 4—figure supplement 4. Top right corner, average Pearson correlation coefficient (PCC) between the two channels; for statistics see Figure 4—figure supplement 5. (B) Change of the syntaxin 1A staining pattern after Stx-full/Stx-?S expression. Staining intensity (region of interest [ROI] intensity), maxima size, and maxima density are related to the average control values (set to 100%). (C) SNAP25 staining pattern after Stx-full/Stx-?S expression. SNAP25 staining intensity (ROI intensity), clustering degree (relative standard deviation of the mean [rSDM]; 100% = 0.47), and maxima density are related to the control. For the relation between rSDM and Stx-full expression level, see Figure 4—figure supplement 6. Values are shown as box plots (n = 6–9 experiments, including three experimental preparations used as well for Figure 2; at least 15 membrane sheets were imaged per condition and experiment), showing the median, the 25th and 75th percentile (box), and the minimum and maximum value. Two-tailed paired Student’s t-test compare (1) control to Stx-full/Stx-?S and (2) Stx-full to Stx-?S; ns = not significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. After exchanging the spectral properties of the labels using ATTO647 for the syntaxin and ATTO594 for the SNAP25 channel, the same trends are observed. The syntaxin 1A maxima are sharper and more defined than SNAP25 maxima as well (Figure 4—figure supplement 7).

Fig 3: Interaction between SNAP25 and Stx-full/Stx-?S.HepG2 cells that do not endogenously express syntaxin 1A and SNAP25 express GFP-SNAP25 alone (control) or together with Stx-full/Stx-?S. For immunoprecipitation of GFP-SNAP25, cell lysate is incubated with anti-GFP agarose beads. Left, Western Blot detecting Stx-full/Stx-?S using an anti-Myc-tag antibody (aMyc) and GFP-SNAP25 using an anti-GFP antibody (aGFP) in the input (cell lysate), flow-through and pull-down. Right, band quantification of pull-down. The aMyc-band intensity is related to the aGFP-band intensity and Stx-full is set to 100 %. Values are given as means ± standard deviation (SD; n = 3 experiments). Two-tailed paired t-test compares Stx-full to Stx-?S; ***p < 0.001. Figure 3—source data 1.Raw Western Blot images and annotated full blot for Figure 3.

Fig 4: Investigation of the extra-synaptic populations of SNAP-25 and Syntaxin 1A in hippocampal neurons upon electrical stimulation. Cultured neurons were co-stained with nanobodies directly coupled to Atto647N and with anti-Synaptophysin antibodies (detected with a secondary coupled to Alexa488) to identify synaptic regions. (a) STED images of the nanobody immunostainings were analyzed. The distribution of extra-synaptic SNAP-25 remains unchanged upon stimulation at 20 Hertz for 60 seconds, resembling the pattern previously observed in Figure 5. For control, stimulation and recovery conditions, 356, 388 and 424 synapse line profiles were analyzed from three independent experiments. (b) Upon stimulation, the population of Syntaxin 1A that is outside of synapses relocates into neighboring synapses. This population recovers if the neurons are further incubated for five minutes at 37 °C after stimulation. For control, stimulation and recovery conditions, 288, 144 and 280 synapses were analyzed from three independent experiments. The exemplary images show the nanobody signals in regions centered on synapses (determined by Synaptophysin staining). The scale bars represent 1 µm.

Fig 5: Rescue of palmitoylation of key HIP14 in substrates in the Hip14-/- mouse.Rescue of palmitoylation deficits of key HIP14 substrates in the Hip14-/- mouse, as assessed by Biotin BMCC assay on HIP14 substrates PSD-95 (a) and SNAP-25 (b). ANOVA p = -0.007, n = 5 each. *p<0.05.