Journal: bioRxiv

Article Title: Nanobinders for Synaptotagmin 1 enable the analysis of synaptic vesicle dynamics in rodent and human models

doi: 10.1101/2025.04.16.649111

Figure Lengend Snippet: a-c ) The NbLumSyt1-APEX2 allows efficient biotinylation of proteins in the proximity of Syt1 upon live uptake in hippocampal neurons to facilitate live-cell proteomic mapping. Representative images of neurons upon uptake of the NbLumSyt1-APEX2 where biotinylated proteins are revealed with fluorescent Streptavidin in a . In the absence of H 2 O 2 , only the few endogenous biotinylated proteins are observable. In the presence of all components, the reaction occurs efficiently, as also revealed by western blot of labeled neurons ( b ). To identify the interactors of Syt1, in situ proximity labelling was performed with the NbLumSyt1-APEX2 ( c ). The electron microscopy image in the scheme is an example of the labelled vesicles, as reveled upon photoconverting 3,3-diaminobenzidine (DAB) into a stable, electron microscopically visible dark product. d ) Protein intensities measured with LC-MS/MS in input and upon enrichment of the biotinylated proteins. For controls neurons without nanobody or neurons where the anti-ALFA-Nb was provided in the medium of neurons are used. Note that, since the primary neurons do not express the ALFA-tag, this control will allow to reveal the effect of the unspecific biotinylation of the membranes occurring during the labelling period. Note that Syt1, as expected, is efficiently biotinylated and enriched upon IP with streptavidin beads. For details concerning this type of experiments and the respective analysis, refer to the methods. e ) Summary of the gene ontologies (GOs; cellular components) for the proteins biotinylated upon live uptake of the NbLumSyt1-APEX2 (for a detailed list see Supplementary Table 1 ). As expected, synaptic components and membrane GOs are over-represented. f ) Possible interactors identified upon live-cell proteomic mapping and enrichment vs. input and vs IP control. Cntfr was found to be the most enriched candidate, together with other proteins that could be studied in future works. As an additional interesting candidate, the integral membrane protein 2B (Itm2b) stands out since it plays a role in vesicle trafficking, is linked to neurodegenerative diseases involving synaptic dysfunction, and may regulate SV recycling and neurotransmitter release . g ) Super-resolution stimulation emission depletion (STED) imaging reveals that ∼20% of boutons labeled with live uptake are also positive for Cntfr. Note that in this case, for cross-validation purposes, the live uptake was performed with the Syt1-luminal antibody. h ) Proximity ligation assay (in situ PLA), using antibodies against the luminal portion of Syt1 and anti-Cntfr, confirms close proximity for these two proteins. As a control, a primary antibody against a protein not expressed in hippocampal neurons (Ribeye) was used. i ) Blocking the network activity of primary hippocampal neurons with tetrodotoxin (TTX) decreases the in situ PLA signal between Syt1 and Cntfr. Stimulation with the ligand of Cntfr (Cntf; 8 nM) does not change the PLA signal between Syt1 and Cntfr. j-k ) Stimulation of neurons with Cntf, increases SV exo-endocytosis following 24h incubation.

Article Snippet: At 14 days in vitro (DIV) neurons were incubated either in conditioned medium only (control), or in conditioned medium supplemented with 8 nM ciliary neurotrophic factor (CNTF, C-245 Alomone Labs) for 2 hours or 24 hours, or 3 µM tetrodotoxin (TTX, 1069 Tocris) for 2 hours.

Techniques: Western Blot, Labeling, In Situ, Electron Microscopy, Liquid Chromatography with Mass Spectroscopy, Control, Membrane, Imaging, Proximity Ligation Assay, Blocking Assay, Activity Assay, Incubation