Journal: bioRxiv
Article Title: Context-dependent Interactors Regulate TDP-43 Dysfunction in ALS/FTLD
doi: 10.1101/2025.04.07.646890
Figure Lengend Snippet: APEX2 proximity labeling and DIA mass spectrometry enable quantitative comparison of context-dependent TDP-43’s interactors. (A) Schematic diagram of the CUTS (CFTR-UNC13A TDP-43 Sensor) biosensor. CUTS biosensor was designed to detect TDP-43 loss of function in splicing by expressing nucleus GFP signal (EGFP-3XNLS) proportional to the level of TDP-43 LOF in real-time. (B) Relative GFP fluorescence intensity quantification from live confocal imaging in stable HEK293 cells expressing doxycycline-inducible (72 h of 1 mg/mL doxycycline) CUTS with the transfection of pCMV-APEX2 backbone-only, TDP-43 WT , TDP-43 ΔNLS , TDP-43 5FL , TDP- 43 5FL/ΔNLS with pCMV-APEX2 backbone or non-transfected (72 h, N=3 biological replicates). (C) Relative GFP fluorescence intensity quantification from live confocal imaging in Hela TDP- 43-KO cells with the co-transfection of doxycycline-inducible (72h of 1 mg/mL doxycycline) CUTS plasmid and pCMV-APEX2 backbone-only, TDP-43 WT , TDP-43 ΔNLS , TDP-43 5FL , TDP- 43 5FL/ΔNLS with pCMV-APEX2 backbone or no plasmid (72 h, N=3 biological replicates). For (B) and (C), statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison test (* = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001). Data are the mean ± s.d. (D) Relative GFP fluorescence intensity quantification from live confocal imaging in stable HEK293 cells expressing doxycycline-inducible (72 h of 1 mg/mL doxycycline) CUTS with the treatment of NaAsO2 (2 h, gradient doses: 0, 67.5, 125, 250, 500, 1000 μM), Sorbitol (4 h, gradient doses: 0, 100, 200, 400, 800, 1600 mM), KCl (4 h, gradient doses: 0, 12.5, 25, 50, 100, 200 mM), Puromycin (24 h, gradient doses: 0, 1.25, 2.5, 5, 10, 20 μg/mL), MG132 (24 h, gradient doses: 0, 5, 10, 20, 40, 80 μM), and Tunicamycin (24 h, gradient doses: 0, 5, 10, 20, 40, 80 μM) (N=2 biological replicates). Data are the mean ± s.d. (E) Schematic diagram of APEX2-TDP-43 system. HEK293 stable cell line with doxycycline- inducible TDP-43 WT , TDP-43 ΔNLS , TDP-43 5FL , or TDP-43 5FL/ΔNLS were generated. BP and H2O2 were added sequentially to enable proximity labeling. (F) Representative immunofluorescence staining images of the biotin signal labeled by APEX2- TDP-43 variants in stable HEK293 cell lines (48 h of 1 mg/mL doxycycline) with or without NaAsO2 stress (250 μM, 2 h). Blue = Hoechst; Red = Streptavidin-Cy3. Scale bar = 2 µm. (G) Schematic diagram of the experimental design of the proximity labeling for data-independent acquisition (DIA) quantitative mass spectrometry with the combination settings of TDP-43 mislocalization, impaired RNA binding, and NaAsO2 stress. APEX2-3XNLS and APEX2-3XNES were included as compartment background controls. N=4 independent biological replicates for each one of the settings. (H) Principal component analysis (PCA) of the raw interactome lists from the seven experiment groups of APEX2-TDP-43 proximity labeling. PC1 (49.94% variance) and PC3 (5.72% variance) are shown as they distinguish different groups. (I) Venn diagram of the proteins detected and quantified by all the groups of APEX2-TDP-43 in this study with the combination of previously reported TDP-43 interactors. (J) Venn diagram of the proteins enriched in APEX2-TDP-43 WT group with previously reported TDP-43 WT (mainly in the nucleus) interactors. (K) Venn diagram of the proteins enriched in APEX2-TDP-43 ΔNLS group with previously reported TDP-43 ΔNLS (mainly in the cytoplasm) interactors. (L) Overlapping diagram of the enriched protein interactors among TDP-43 WT , TDP-43 5FL , and TDP-43 WT +AS groups. (M) Overlapping diagram of the enriched protein interactors among TDP-43 ΔNLS , TDP-43 5FL/ΔNLS , TDP-43 ΔNLS +AS, and TDP-43 5FL/ΔNLS +AS groups. For (J) to (M), protein interactors from each group are enriched with log2FC > 0.3 and P < 0.05, comparing with their respective compartment controls (APEX2-3XNLS for the nucleus, APEX2- 3XNES for the cytoplasm).
Article Snippet: For the enrichment of biotinylated proteins, 15% of the cell lysates from one 10-cm dish were diluted five times with RIPA buffer and incubated with 50 μL NanoLINK Streptavidin Magnetic Beads (TriLink Biotechnologies, M-1002) overnight at 4°C on a rotator.
Techniques: Labeling, Mass Spectrometry, Comparison, Expressing, Fluorescence, Imaging, Transfection, Cotransfection, Plasmid Preparation, Stable Transfection, Generated, Immunofluorescence, Staining, Data-independent acquisition, RNA Binding Assay
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