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: Development and characterization of a nanobody against the luminal domain of the calcium sensor Synaptotagmin 1. a ) Schematic representation of nanobody selection and characterization. b ) Immunofluorescence images of live-labeled hippocampal neurons with NbLumSyt1 fused to the Halo-Tag. This tool can be used to label actively recycled synaptic vesicles and provides excellent signal-to-noise images. c ) In live imaging and retrospective immunofluorescence, NbLumSyt1 colocalizes with the presynaptic scaffold protein Bassoon and labels synaptic boutons, including excitatory (VGLUT + ) and inhibitory (VGAT + ) boutons. d ) Western blot analysis of purified Syt1. The nanobody can be used in WB applications and recognizes increasing concentrations of purified Syt1. e ) Syt1 immunoprecipitation in WT or Syt1 KO mouse primary neurons using the nanobody, followed by western blot using an Syt1 antibody. Note that the smear-like effect in the IP for the WT samples is likely due to gel overloading. f ) Immunofluorescence imaging following nanobody uptake in WT and Syt1 KO primary neurons and post-fixation staining of VGLUT. g ) A peptide microarray binding assay with synthesized and immobilized rat and human Syt1 protein sequences identified the region of Syt1 that the nanobody binds to in both species. h ) Crystal structure of the nanobody (orange) bound to Syt1 (blue). i ) Specific amino acids involved in nanobody-Syt1 binding (PDB: 8B8I). Scale bars: 10 μm in b , c , and f -inset; 30 μm in f . The error bars indicate the means ± SEMs for panels e and g and the 5th to 95th percentiles for the boxplot in panel f; ns, not significant, **** p < 0.0001

Article Snippet: The samples were loaded on an SDS gel, and western blotting was performed using Syt1 cytoplasmic (SynapticSystem, cat. #105 011, AB_887832) and GAPDH (ThermoScientificTM, cat. MA5–15738, RRID: AB_10977387) antibodies.

Techniques: Selection, Immunofluorescence, Labeling, Imaging, Western Blot, Purification, Immunoprecipitation, Staining, Peptide Microarray, Binding Assay, Synthesized

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: NbLumSyt1 is a minimally invasive tool for measuring synaptic vesicle exo-endocytosis. a-c ) Miniature excitatory postsynaptic current (mEPSC) measurements in control (no nanobody or antibody treatment) and treated neurons with different loading times, either with NbLumSyt1 directly fused with pHluorin (NbLumSyt1-pHluorin) or with a commercial Syt1-luminal antibody (SySy 604.2). d ) Live imaging of NbLumSyt1-pHluorin recycling. The fluorescence increase indicated nanobody uptake upon stimulation, followed by florescence decay due to endocytosis of the nanobody. Treatment with ammonium chloride (NH 4 + ) revealed the total pool of labeled vesicles. Scale bar: 10 μm. e ) Example traces with decay time constants (tau) and amplitudes upon electrical stimulation of cells labeled with NbLumSyt1-pHluorin. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test. Although there is a trend, no significant differences were found. f ) Percentage of boutons responding to different electrical stimuli (calculated as a percentage of the total pool of SVs, as defined by NH 4 + treatment). g ) Evoked inhibitory postsynaptic currents (eIPSCs), which are used to study the drive of GABAergic neurotransmission. Control neurons were stimulated with 45 mM KCl for 5 min without the nanobody. Inset: Quantification of eIPSC amplitude. h ) Comparison of the effects of NbLumSyt1-pHluorin on neurons overexpressing Syt1 pHluorin. The overexpression of Syt1-pHluorin causes slower endocytosis at higher (40 Hz) stimulation frequency. i ) Evaluation of the surface fraction by perfusion with acidic buffer (pH 5). Ammonium chloride perfusion at the end of the experiment was used to reveal the total population of molecules (used for normalization). The surface fraction was unchanged at our Syt1-pHluorin expression levels. j -m) The fluorescence increase and recovery in neurons loaded with NbLumSyt1-TEV-mOrange2, incubated with the TEV protease, and then field-stimulated either for 2 s at 20 Hz ( j ) or for 10 s at 30 Hz (l) in the presence or absence of the reacidification blocker bafilomycin. Under control conditions, as expected, fluorescence is quenched and returns to baseline after endocytosis due to acidification of the endocytosed Syt1 pools. After mild stimulation ( j , recovery at 110 s quantified in k ), the fluorescence in TEV-cleaved neurons does not recover; but in contrast, after more pronounced stimulation, fluorescence quenching is observed ( l , recovery at 110 s quantified in m ). The graphs summarize the results of 3 independent experiments. The error bars indicate the means ± SEMs; ns, not significant, * p < 0.05, *** p < 0.001,**** p < 0.0001

Article Snippet: The samples were loaded on an SDS gel, and western blotting was performed using Syt1 cytoplasmic (SynapticSystem, cat. #105 011, AB_887832) and GAPDH (ThermoScientificTM, cat. MA5–15738, RRID: AB_10977387) antibodies.

Techniques: Control, Imaging, Fluorescence, Labeling, Comparison, Over Expression, Expressing, Incubation

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: The samples were loaded on an SDS gel, and western blotting was performed using Syt1 cytoplasmic (SynapticSystem, cat. #105 011, AB_887832) and GAPDH (ThermoScientificTM, cat. MA5–15738, RRID: AB_10977387) antibodies.

Techniques: Western Blot, Labeling, In Situ, Electron Microscopy, Control, Membrane, Imaging, Biomarker Discovery, Proximity Ligation Assay, Negative Control, Blocking Assay, Activity Assay

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: Live-cell single-molecule imaging of endogenous Syt1 reveals two populations of molecules whose displacement dynamics differ. a ) Scheme of single fluorescent molecule detection in live hippocampal neurons via uPAINT. To detect and track single molecules, cells were incubated with complexes conjugated prior to imaging the NbLumSyt1-Halo-Tag with the Halo-ligand JF549 or the NbLumSyt1-pHluorin with the anti-GFP Nb-At647N (NbLumSyt1-pH/Nb-At647N). Individual trajectories correspond to probe-binding events and subsequent lateral diffusion of surface-exposed Syt1 molecules and do not represent the full exocytosis–endocytosis cycle of individual synaptic vesicles. b) Super-resolved images of single-molecule trajectories, diffusion coefficients, and average intensities in hippocampal neurons over 16,000 frames. The color scale in the diffusion coefficient map ranges from 0 to 1, corresponding to Log10 diffusion coefficient detections, and the colder colors in the scale indicate lower mobility. The average intensity map represents localization densities as arbitrary units, with warmer colors indicating higher detection densities. c-g) Quantification of the parameters under different conditions. The lifetime decreases with increasing complex size, and the overexpression of Syt1 results in a significantly greater number of trajectories than do the endogenous Syt1 trajectories. h) Representative image showing detection of the NbLumSyt1-Halo/JF549 complex from a 320 s acquisition at 50 Hz by imaging with uPAINT in cultured hippocampal neurons. The arrowheads indicate two separate NbLumSyt1-Halo/JF549 clusters, and the boxed areas (i-iii) are magnified. The resulting NASTIC analysis images of 2D kernel density estimation (KDE) of detections with single-molecule trajectories, centroids, clusters, and instantaneous diffusion coefficients (log10) from the boxed region are shown magnified in the boxed regions. The trajectories are shown in gray, with each trajectory segment representing the displacement of the detected molecule in 0.02 s. The centroid of each trajectory is indicated with a red dot. The colored convex hulls indicate the extent of the detections associated with clustered trajectories as determined by NASTIC. i) The top panel shows the 2D temporal clustering NbLumSyt1-Halo/JF549 (arrow and arrowhead point to the timeframe of blue and green clusters shown in h) over 320 s, and the lower panel shows the respective diffusion coefficient mobility of the trajectories, indicating a lower mobility of NbLumSyt1-Halo/JF549 within clusters than when unclustered. j) 3D representation of the spatiotemporal clustering of NbLumSyt1-Halo/JF549, where the blue and green clusters from c and f are indicated. k) The graph shows the quantification of the mean square displacement (µm2 s− 1) of NbLumSyt1-Halo/JF549 within clusters and outside of the clusters (unclustered). MSD curves measure the average mobility with respect to time of all the trajectories observed. l) Biophysical properties of NbLumSyt1-Halo/JF549 clusters (n = 10 neurons). The numbers represent average values ± SEMs. Scale bars: 1 μm. Statistical analysis of normally distributed data was performed via one-way ANOVA for multiple comparisons in g, and for nonnormally distributed data, one-way ANOVA with the Kruskal‒Wallis test for multiple comparisons was used. ** p < 0.01, **** p < 0.0001.

Article Snippet: The samples were loaded on an SDS gel, and western blotting was performed using Syt1 cytoplasmic (SynapticSystem, cat. #105 011, AB_887832) and GAPDH (ThermoScientificTM, cat. MA5–15738, RRID: AB_10977387) antibodies.

Techniques: Imaging, Incubation, Binding Assay, Diffusion-based Assay, Over Expression, Cell Culture

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: NbLumSyt1 specifically binds human Syt1 and can be used for recycling assays in human neurons. a ) Human iPSC-derived hypothalamic-like neurons were stained live with NbLumSyt1 and, after recycling, fixed and immunoassayed against endogenous Syt1 and the neuron-specific betaIII tubulin marker (TuJ). The cells were pretreated with TTX or K + during labeling. b ) Quantification indicates that increased stimulation during labeling increases both the labeling level (upper panel) and the correlation between luminal Syt1 and total Syt1 (lower panel). Unpaired Student’s t test; the error bars indicate the means ± SEMs; ** p < 0.01, **** p < 0.0001. c ) Synaptic vesicle recycling measured in iNeurons monitored with the pH-sensitive version of NbLumSyt1. (Left) Averaged normalized pHluorin fluorescence traces from iNeurons (6–8 weeks in culture, N = 3) stimulated with 200 action potentials (40 Hz, 5 s) at physiological temperature (37 °C). The blue segmented line indicates the beginning of the stimulation. (Right) Representative images showing the sensor intensity before (t = 0 s) and directly after (t = 16 s) electrical stimulation. Scale bars: 10 μm in a ; 5 μm in c

Article Snippet: The samples were loaded on an SDS gel, and western blotting was performed using Syt1 cytoplasmic (SynapticSystem, cat. #105 011, AB_887832) and GAPDH (ThermoScientificTM, cat. MA5–15738, RRID: AB_10977387) antibodies.

Techniques: Derivative Assay, Staining, Marker, Labeling, Fluorescence

Journal: Cell Reports Medicine

Article Title: A bispecific nanobody-drug conjugate targeting TROP2 and c-Met for low-concentration, single-dose treatment of pancreatic cancer

doi: 10.1016/j.xcrm.2026.102688

Figure Lengend Snippet: Expression profiles of TROP2 and c-Met in tumor and normal tissues (A) TROP2 gene expression profile in pancreatic tumor and normal tissues in humans. See also . (B) MET gene expression profile in pancreatic tumor and normal tissues in humans based on data from GEPIA (Gene Expression Profiling Interactive Analysis). (C) Comparative analysis of TROP2 and MET gene expression in tumor tissues and normal tissues in pancreatic cancer (PAAD), lung adenocarcinoma (LUAD), and colon cancer (COAD) patients. See also . (D) Overall survival analysis of patients with high versus low TROP2 or MET gene expression. (E) Multicolor fluorescence tissue microarray analysis of TROP2 and c-Met protein expression in tumor tissues from 55 clinical patients (spanning seven cancer types) and normal tissues. Quantitative fluorescence analysis was performed, with TROP2 shown in red and c-Met shown in green. (F) Quantitative fluorescence distribution of TROP2 and c-Met protein expression from (E). (G) Representative multicolor fluorescence images of tissue microarrays from PAAD, LUAD, and COAD patients, showing TROP2 (red), c-Met (green), and nuclei (blue, DAPI). Scale bars, 200 μm and 100 μm. See also . (H) Flow cytometry analysis of TROP2 and c-Met expression in representative cell lines derived from different cancer types, including BxPC-3, NCI-H358, SPC-A1, MDA-MB-231, and HT-29 ( n = 3 independent experiments). See also .

Article Snippet: c-Met (Cytoplasmic) Polyclonal antibody , Wuhan Sanying , Cat#25869-1-AP; RRID: AB_2880276.

Techniques: Expressing, Gene Expression, Fluorescence, Microarray, Flow Cytometry, Derivative Assay

Journal: Cell Reports Medicine

Article Title: A bispecific nanobody-drug conjugate targeting TROP2 and c-Met for low-concentration, single-dose treatment of pancreatic cancer

doi: 10.1016/j.xcrm.2026.102688

Figure Lengend Snippet: Development and characterization of nanobody-based BsADCs (A) Structural design of four bispecific antibodies targeting TROP2 and c-Met (created with BioRender.com ). (B) Schematic representation of the B6ADC structure. (C) Determination of the DAR of reduced B6ADC single chains via LC-MS ( n = 3 independent experiments). (D) Binding affinities of B6HCAb and B6ADC for the TROP2 and c-Met proteins, measured by Biacore 8K ( n = 3 independent experiments). (E) Schematic and results validating the ability of the B6HCAb-1 to simultaneously bind TROP2 and c-Met antigens ( n = 3 independent experiments). Scale bar, 10 μm. (F) AlphaFold3-predicted key hydrogen bonding interaction sites between the TROP2 VHH and TROP2 proteins ( n = 3 independent experiments). See also . (G) AlphaFold3-predicted key hydrogen bonding interaction sites between the c-Met VHH and the c-Met protein ( n = 3 independent experiments). See also . (H) Binding capacity of B6HCAb to TROP2 antigen and its variants ( n = 2∼3/group). (I) Binding capacity of B6HCAb to c-Met antigen and its variants ( n = 2∼3/group). See also .

Article Snippet: c-Met (Cytoplasmic) Polyclonal antibody , Wuhan Sanying , Cat#25869-1-AP; RRID: AB_2880276.

Techniques: Liquid Chromatography with Mass Spectroscopy, Binding Assay

Journal: Cell Reports Medicine

Article Title: A bispecific nanobody-drug conjugate targeting TROP2 and c-Met for low-concentration, single-dose treatment of pancreatic cancer

doi: 10.1016/j.xcrm.2026.102688

Figure Lengend Snippet: Validation of the mechanism and cytotoxicity of B6ADC (A and B) Colocalization of B6ADC with lysosomes. B6ADC is shown in red, lysosomes are shown in green, and nuclei are shown in blue (DAPI) ( n = 3 independent experiments). Scale bars, 20 μm and 10 μm. (C and D) Penetration ability of B6ADC and parental ADCs in BxPC-3 tumor spheroids. B6ADC is shown in red, and nuclei are shown in blue (DAPI) ( n = 3 independent experiments). Scale bar, 100 μm. See also . (E) Cytotoxicity of B6ADC and parental ADCs, including BxPC-3, NCI-H358, SPC-A1, MDA-MB-231, HT-29, and OVCAR-3, in various tumor cell lines with different TROP2/c-Met expression levels ( n = 3/group). See also and . (F) Cytotoxicity of B6ADC to normal human cells (HUVECs and MRC-5 cells) ( n = 3/group). (G) Tumor spheroid lysis ability of B6ADC and parental ADCs in BxPC-3 tumor spheroids ( n = 3 independent experiments). Scale bar, 100 μm. Statistical analysis was performed using two-way ANOVA. The data are presented as the means ± SDs. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and p > 0.05 (no significance, ns).

Article Snippet: c-Met (Cytoplasmic) Polyclonal antibody , Wuhan Sanying , Cat#25869-1-AP; RRID: AB_2880276.

Techniques: Biomarker Discovery, Expressing, Lysis

Journal: Cell Reports Medicine

Article Title: A bispecific nanobody-drug conjugate targeting TROP2 and c-Met for low-concentration, single-dose treatment of pancreatic cancer

doi: 10.1016/j.xcrm.2026.102688

Figure Lengend Snippet: Broad-spectrum antitumor activity of B6ADC (A) Tumor suppression efficacy of B6ADC in multiple pancreatic cancer CDX models ( n = 5∼7/group). (B) Tumor suppression efficacy of B6ADC in four cell lines with varying TROP2/c-Met expression levels ( n = 5∼7/group). Scale bar, 50 μm. (C) Tumor suppression effect of a single 2.2 mg/kg dose of B6ADC in SPC-A1 lung adenocarcinoma-bearing mice with a tumor volume of 1,200 mm 3 ( n = 5∼7/group). (D) Comparison of tumor suppression between B6ADC and the combination of parental “TROP2ADC and c-MetADC” in SPC-A1 tumor-bearing mice (initial tumor volume: 600 mm 3 ) ( n = 6/group). See also . (E) Comparison of tumor suppression between B6ADC and the combination of “marketed TROP2ADC (SG) and marketed c-MetADC (Teliso-V)” in SPC-A1 tumor-bearing mice (initial tumor volume: 600 mm 3 ) ( n = 5∼7/group). See also . (F) Transmission electron microscopy images showing apoptosis in BxPC-3 tumors on day 7 after treatment with B6ADC and the control. Apoptotic features (e.g., chromatin condensation and nuclear fragmentation) were observed in the B6ADC-treated group ( n = 3 independent experiments). Scale bar, 2 μm. See also . Statistical analysis was performed using two-way ANOVA. The data are presented as the means ± SDs. ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, and p > 0.05 (no significance, ns).

Article Snippet: c-Met (Cytoplasmic) Polyclonal antibody , Wuhan Sanying , Cat#25869-1-AP; RRID: AB_2880276.

Techniques: Activity Assay, Expressing, Comparison, Transmission Assay, Electron Microscopy, Control

Journal: Cell Reports Medicine

Article Title: A bispecific nanobody-drug conjugate targeting TROP2 and c-Met for low-concentration, single-dose treatment of pancreatic cancer

doi: 10.1016/j.xcrm.2026.102688

Figure Lengend Snippet: Safety and pharmacokinetics of B6ADC (A) Body weight changes in BALB/c mice treated with B6ADC at doses of 44 mg/kg, 66 mg/kg, and 110 mg/kg ( n = 4∼6/group). See also . (B) Dose-dependent safety evaluation of B6ADC in TROP2-humanized mice and c-Met-humanized mice ( n = 4/group). (C) Biochemical analysis of liver function (AST, ALT, and T-Bil) and kidney function (Cr and BUN) in TROP2-humanized mice and c-Met-humanized mice ( n = 4/group). (D) MMAE release level of B6ADC in BALB/c mice ( n = 5/group). (E) MMAE release in tumor tissues, serum, and major organs (heart, liver, spleen, lung, and kidney) at different time points after the administration of 2.2 mg/kg B6ADC to BALB/c mice ( n = 3/group). Statistical analysis was performed using two-way ANOVA. The data are presented as the means ± SDs. ∗∗∗∗ p < 0.0001.

Article Snippet: c-Met (Cytoplasmic) Polyclonal antibody , Wuhan Sanying , Cat#25869-1-AP; RRID: AB_2880276.

Techniques: Drug discovery