Journal: bioRxiv

Article Title: Novel single molecule imaging approaches reveal structure-function alterations to the nuclear pore complex in early C9ORF72 -associated TDP-43 proteinopathy

doi: 10.64898/2026.02.17.706337

Figure Lengend Snippet: Neuroblastoma cell line SH-SY5Y untreated (control) or with acute treatment (1hr) of polypeptide GR20 (10µM), as per single molecule tracking experiments, and then immunostained with dye-conjugated antibodies for the NPC components Nup98 and Nup50. a) Example fluorescence image of improved immunostaining detection of individual NPC spot detection of AF647-labelled Nup50 (magenta) and AF488-labelled Nup98 (green) acquired using HILO microscopy, with enlarged view and respective definition of Nup50/98 positions; scale bars are 5 μm and 500nm for enlarged views. b) Representative fluorescence images of untreated (control) and polyGR-treated cells with output spot detection for Nup98 and Nup50 within deep learning nuclear envelope central boundary definition (yellow dashed line); scale bars are 5 μm. c) Schematics of process for paired particle localisation and alignment averaging for Nup98 positioning (green) in reference to Nup50 (magenta). d) Output density heatmap of Nup98 positioning in reference to Nup50 from paired localisation analysis for experimental data of control and polyGR-treated cells with comparative best fit simulation heatmap, with values for mean radii – here, indicating the distance between Nup50 and Nup98 - (±SD) and structural fidelity between experimental and best fit simulation data using the structural similarity index (SSIM) below. e) Best fit simulated distribution of Nup50 (magenta) and Nup98 (green) – now reflecting their effective position in a nuclear pore complex - used to generate heat map in (d) with lines for respective median Nup98 and Nup50 diameter in control and polyGR treated conditions. (f) Quantification for determined median diameter (±95% CI) of Nup98 and Nup50 under control and polyGR-treated conditions using paired localisation and diameter simulation analysis. Statistical testing: unpaired Mann Whitney; ****p<0.0001; N>250; raw numerical data in Source Data File for Fig. 3.

Article Snippet: Cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour at room temperature, then incubated overnight at 4°C with conjugated antibodies (1:50) freshly diluted in blocking buffer, including anti-Nup50 (G-4) Alexa Fluor 647–conjugated antibody (sc-398993 AF647, Santa Cruz Biotechnology) and anti-Nup98 (C-5) Alexa Fluor 488–conjugated antibody (sc-74578 AF488, Santa Cruz Biotechnology).

Techniques: Control, Fluorescence, Immunostaining, Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Novel single molecule imaging approaches reveal structure-function alterations to the nuclear pore complex in early C9ORF72 -associated TDP-43 proteinopathy

doi: 10.64898/2026.02.17.706337

Figure Lengend Snippet: Nucleocytoplasmic transport (beige) in physiological and C9ORF72 polyGR (10 µM) disease conditions with width and density of arrow denote transport passage and molecular density, and percentage success rate of tracks. Nuclear pore structure schematic in grey in cross section (upper) and nuclear side view (lower) with Nup98 positions denoted in green and Nup50 in magenta, arrows highlighting change in organisation upon exposure to polyGR, and values for simulated diameter measurement. Associated change in nucleocytoplasmic distribution of disease-associated protein TDP-43 shown in blue. Created in BioRender.

Article Snippet: Cells were washed three times with PBS and blocked with 5% BSA in PBS for 1 hour at room temperature, then incubated overnight at 4°C with conjugated antibodies (1:50) freshly diluted in blocking buffer, including anti-Nup50 (G-4) Alexa Fluor 647–conjugated antibody (sc-398993 AF647, Santa Cruz Biotechnology) and anti-Nup98 (C-5) Alexa Fluor 488–conjugated antibody (sc-74578 AF488, Santa Cruz Biotechnology).

Techniques:

Journal: bioRxiv

Article Title: Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

doi: 10.64898/2026.01.18.700178

Figure Lengend Snippet: (a) The qExM framework targets endogenous proteins or protein complexes using pairs of antibodies against non-overlapping epitopes, leveraging western blot-effective antibodies enabled by expansion microscopy’s improved labeling efficiency. (b) Schematic representation of the labeling outcome categories in a qExM experiment. The total population of targets can be labeled by either antibody A (blue), antibody B (red), both antibodies (dual-labeled), or remain unlabeled. Each label provides a random subset of the total population, allowing assessment of the other label’s efficiency. (c) Mathematical formulation of the estimators used in qExM to calculate labeling efficiency of each antibody and the total abundance of target proteins, derived from the Chapman capture-recapture method. (d , e , f) Fluorescence microscopy images of U2OS cells prepared using three expansion microscopy protocols and collected and displayed with identical parameters: d: chemical fixation with glycidyl methacrylate anchoring (ChemGMA), e: cryo-fixation with formaldehyde/acrylamide anchoring (CryoFAA), and f: cryo-fixation with glycidyl methacrylate anchoring (CryoGMA). Cells were immunolabeled for Nup96-GFP (cyan), mitochondrial MT-CO1 (magenta), and α-tubulin (yellow). Scale bar 5 µm. (g) Signal to Noise measurements comparing the mitochondrial MT-CO1 signal across the three expansion microscopy protocols.

Article Snippet: In this study we used the following primary antibodies: for CI: NDUFS2 (Abcam ab192022), ND2 (Sigma MABS2047), for CIII: UQCRCP1 (Abcam ab14746), UQCRFS1/RISP (Abcam ab11925), for CIV: MT-CO1 (Abcam ab14705), MT-CO2 (Abcam ab79393), GFP (Abcam ab6673), Nup96 (ProteinTech-12329-1-AP), Nup54 (Sigma HPA035929), ELYS (Sigma HPA031658), P5CS (Proteintech 17719-1-AP, 68184-1-IG) All antibodies were used at a dilution of 1:100, except ELYS which was used at a concentration of 1:50.

Techniques: Western Blot, Labeling, Formulation, Derivative Assay, Fluorescence, Microscopy, Immunolabeling

Journal: bioRxiv

Article Title: Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

doi: 10.64898/2026.01.18.700178

Figure Lengend Snippet: (a) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and Nup96 (blue) labeling. (b) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and Nup54 (blue) labeling. (c) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and ELYS (blue) labeling. Smaller images in a-c show individual nuclear pores at higher magnification, demonstrating the characteristic ring-like arrangement of NPC subunits. (d) Structural model of the human nuclear pore complex highlighting the positions of Nup96-mEGFP (green), Nup54 (yellow), and ELYS (magenta) within the 8-fold symmetrical arrangement. (e) Image analysis workflow showing raw data (Image), segmentation mask (Segmentation), instance identification (Centers), and colocalization analysis (Colocalization). (f) Labeling efficiency estimates from qExM mEGFP and each target protein. “Direct Counting” refers to the labeling efficiency defined by the number of observed target per nuclear pore, knowing that there should be 8 total targets. No statistical significance was found between groups (mEGFP p = 0.23, Nup96 p= 0.65, Nup54 p= 0.28, ELYS p = 0.19) (g) Estimated abundance of protein subunits for each experiment, showing means of 7.4, 7.0, and 8.7 for Nup96, Nup54, and ELYS, respectively, closely matching the expected 8-fold symmetry. Scale bars: 500 nm (overview images) and 50 nm (insets). 3 experimental replicates were made for each imaging pair. 45 images of 1365 NPC wlabeled ith Nup96-mEGFP with Nup96. 41 images of 1465 NPC labeled with Nup96-mEGFP with Nup54. 32 images of 1118 NPC labeled with Nup96-mEGFP with ELYS.

Article Snippet: In this study we used the following primary antibodies: for CI: NDUFS2 (Abcam ab192022), ND2 (Sigma MABS2047), for CIII: UQCRCP1 (Abcam ab14746), UQCRFS1/RISP (Abcam ab11925), for CIV: MT-CO1 (Abcam ab14705), MT-CO2 (Abcam ab79393), GFP (Abcam ab6673), Nup96 (ProteinTech-12329-1-AP), Nup54 (Sigma HPA035929), ELYS (Sigma HPA031658), P5CS (Proteintech 17719-1-AP, 68184-1-IG) All antibodies were used at a dilution of 1:100, except ELYS which was used at a concentration of 1:50.

Techniques: Labeling, Imaging