Journal: Journal of Nanobiotechnology
Article Title: A versatile nanobody platform for live and super-resolution imaging of synaptic vesicle dynamics and plasticity in rodent and human neurons
doi: 10.1186/s12951-026-04489-w
Figure Lengend Snippet: Syt1 is found in close proximity to the ciliary neurotrophic factor receptor (Cntfr) and regulates synaptic vesicle dynamics. a-c ) 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 NbLumSyt1-APEX2, where biotinylated proteins are revealed with fluorescent streptavidin (a) . In the absence of H 2 O 2 , only a few endogenous biotinylated proteins are observable. In the presence of all the components, the reaction occurred efficiently, as revealed in western blot analysis of labeled neurons ( b ). To identify the interactors of Syt1, in situ proximity labeling was performed with NbLumSyt1-APEX2 ( c ). The electron microscopy image in the scheme is an example of the labeled vesicles, as revealed upon photoconverting 3,3’-diaminobenzidine (DAB) into a stable, electron microscopically visible dark product. d ) Protein intensities measured with LC‒MS/MS at the input and upon enrichment of the biotinylated proteins. Two controls were used: neurons without nanobodies or neurons where an unrelated nanobody (anti-ALFA-Nb) was provided in the medium. Note that since the primary neurons do not express the ALFA tag, this control will reveal the effect of the unspecific biotinylation of the membranes occurring during the labeling period. Note that Syt1, as expected, is efficiently biotinylated and enriched upon IP with streptavidin beads. See methods for details concerning the experiments and analyses. e ) Summary of the gene ontologies (GOs; cellular components) for the proteins biotinylated upon live uptake of NbLumSyt1-APEX2 (for a detailed list, see Supplementary Table 1). As expected, synaptic components and membrane GO terms were overrepresented. f ) Possible interactors identified via 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. g ) Super-resolution stimulation emission depletion (STED) imaging reveals that ~ 20% of boutons labeled with live uptake are also positive for Cntfr. In this case, for cross-validation purposes, live uptake was performed with the 604.2 Syt1-luminal antibody. h ) Proximity ligation assay (in situ PLA) using antibodies against the luminal portion of Syt1 and anti-Cntfr confirmed the close proximity of these two proteins. A primary antibody against a protein not expressed in hippocampal neurons (Ribeye) was used as the negative control. i ) Blocking the network activity of primary hippocampal neurons with tetrodotoxin (TTX) for 2 h decreases the in situ PLA signal between Syt1 and Cntfr. Stimulation with the ligand of Cntfr (Cntf; 8 nM) for 2 h does not change the PLA signal between Syt1 and Cntfr. j , k ) Stimulation of neurons with Cntf for 24 h increases SV exo-endocytosis. Scale bars: 10 μm in a ; 500 nm in c ; 5 μm in g-j . The error bars indicate the means ± SEMs for panel d, and the 5th or 95th percentile for box plots; ** p < 0.01; *** p < 0.001
Article Snippet: At 14 days in vitro (DIV), the 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 2–24 h or with 3 μM tetrodotoxin (TTX, 1069 Tocris) for 2 h. As a proxy for neuronal activity, neurons were exposed to an anti-Syt1 luminal primary antibody directly labeled with ATTO647N (1:200, 105 311AT1 clone 604.2 Synaptic Systems) in conditioned medium for 30 min at 37 °C.
Techniques: Western Blot, Labeling, In Situ, Electron Microscopy, Control, Membrane, Imaging, Biomarker Discovery, Proximity Ligation Assay, Negative Control, Blocking Assay, Activity Assay
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