Fig 2: Expression patterns of SNAP25 during development and relationships of the expression pattern of SNAP25, SYT2, and CHAT. (a) and (b) SNAP25 expression was detected in outer hair cells of the basal turn at E96, while no expression was observed in inner hair cells. no expression was observed in spiral ganglion neurons. (c) At E101, SNAP25 expression came to be detected in both inner and outer hair cells. SNAP25 expression was also detected in the neurite around the inner hair cells. (d) At E101, Coexpression of SYT2 and SNAP25 was observed in the organ of Corti. (e) At E115, SNAP25 expression in the hair cells was diminished, and SNAP25 expression in the neurite terminus was more dominantly observed. (f) and (g) At E115, SNAP25 expression was observed in the CHAT-positive efferent nerve. (h) At E115, SYT2 expression was observed in CHAT-positive efferent neurons. (i) At E115, coexpression of the SYT2 and SNAP25 was observed in the organ of Corti. (j) At P0, expression of SNAP25 was observed in both synaptic termini of both afferent and efferent neurons. (k) and (l). At P0, in the organ of Corti, SNAP25 expression was observed in CHAT- positive TSB and CHAT-negative OSB: SNAP25 expression was detected in both efferent and afferent systems. (m) At P0, SYT2 expression was observed in CHAT-positive efferent neurons. The nuclei were counterstained with Hoechst (blue). Scale bar: 50 µm in (a), 20 µm in (b)–(m). SGN: spiral ganglion neuron, OC: organ of Corti, IHC: inner hair cells, OHC: outer hair cells

Fig 3: Overexpression of miR-335 and its effect on the expression of three of its putative protein targets. (A) Expression of miR-335 using mature miR-335 mimic (OE335; black bar) relative to its expression using scramble control (SCR; white bar). Mir-335 expression was normalized to endogenous controls U6 and U87. (n = 4; *P < 0.05). (B, C, D). Average protein levels of SNAP25, STXBP1 and SYT11 in OE335 (black bar) compared to SCR (white bar) cells (n = 4 each group; *P < 0.05). Representative western blots are shown for each protein. Expression levels were normalized to beta-actin or cyclophilin B (PPIB).

Fig 4: BoNT/C1 ad co-localizes with synaptic markers after neuronal internalization.Cultures of 14-DIV rat hippocampal neurons were exposed to 25 nM BoNT/C1 ad (probed with anti-human IgG 4C10.2) for 16 hours. Cells were prepared for immunocytochemistry, and analyzed using confocal microscopy for the light chain of BoNT/C1 ad (red staining) and for pre-synaptic markers SNAP-25 (A) and VAMP-2 (B), lysosomal marker LAMP-1 (C), and early endosomal marker EEA-1 (D) (green staining). Yellow color in merged images shows co-localization of BoNT/C1 ad LC and the specific marker. Bar = 10 µm.

Fig 5: Model of SNAP25 crowd recruitment to a syntaxin 1A cluster.Syntaxin 1A molecules form tightly packed clusters resembling a bunch of flowers. SNAP25 is enriched in cholesterol-dependent lipid phases (shaded membrane). In the unbound crowd (right), the GFP-tags are able to move freely. Hence, it is less likely that two GFPs are close enough for self-quenching. Moreover, in the unbound crowd all GFPs are accessible to the nanobody. This is different in the bound crowd (left). Here, the N-terminal SNARE domain of SNAP25 interacts with the surface of the syntaxin 1A cluster, positioning the crowd close to the trunk such that some of the GFP-tags are shielded under the cluster’s roof and are no longer accessible to nanobody binding. The GFPs pack more densely and are immobilized, which promotes self-quenching (see grey GFP barrel). One syntaxin 1A cluster can recruit several SNAP25 crowds, explaining how syntaxin controls a surplus of SNAP25. The legend shows the binding probes, GFP-SNAP25 and syntaxin 1A. SNARE domains of SNAP25 are unstructured, unless they participate in an interaction with syntaxin 1A during which they become alpha-helical (in the cartoon alpha-helicity is indicated by drawing the SNARE domain as a rectangle).