Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Co-IP assay to verify the interaction of TDP-43 with PDI in HEK-293T cells transiently co-transfected with TDP-43 and HA-tagged wild-type PDI or HA-tagged dnPDI treated with 1 mM H 2 O 2 for 1 h. Anti-TDP-43 antibody-binding beads were used for co-IP experiments and then detected by western blotting using anti-HA, anti-TDP-43, and anti-β-actin antibodies. ( B and C ) Schemes of TDP-43-PDI constructs used in BiFC assay: TDP-43-EGFP 1-172 fusion protein (B) and signal peptide-HA-EGFP 155-238 -a-b-b’-x-a’-c fusion protein (C). ( D ) Specific interaction between TDP-43 and PDI in living cells was detected by BiFC. HEK-293T cells transiently expressing both TDP-43-EGFP 1-172 and EGFP 155-238 -wild-type PDI or EGFP 155-238 -dnPDI constructs were treated with 1 mM H 2 O 2 for 1 h and cultured for 1 day. Shown are nuclei stained with Hoechst 33342 (blue). EGFP (green) was observed in (D). ( E ) TDP-43 selectively recruits PDI into its phase-separated condensate. Fluorescence images of in vitro phase-separated droplets (red; Merge: yellow) of 10 μM TAMRA-labeled bacterial-purified TDP-43 incubated with Tris-HCl buffer (pH 8.0) containing 10 μM FITC-labeled bacterial-purified wild-type PDI, dnPDI, or SNO-PDI (green) and 10% (w/v) PEG 3350 on ice, and FITC-labeled BSA as a control. ( F ) Regulation of TDP-43 LLPS by PDI. Fluorescence images of 2.5, 5, 7.5, or 10 μM recombinant TDP-43 labeled by TAMRA (red) and incubated with Tris-HCl buffer containing wild-type PDI, dnPDI, or SNO-PDI at 10 μM and 10% PEG 3350 on ice. Scale bars, 10 μm. ( G to J ) The dependence of turbidity changes for LLPS of TDP-43 (G), TDP-43 + 10 μM wild-type PDI (H), TDP-43 + 10 μM dnPDI (I), or TDP-43 + 10 μM SNO-PDI (J) on the concentration of TDP-43 ([TDP-43]) was expressed as mean ± SD (with error bars) of values obtained in three independent experiments. The turbidity of TDP-43 condensates was measured at 600 nm at 25 °C. Representative calculation based on turbidity measurements to determine saturation concentrations of TDP-43 or TDP-43 + dnPDI (open circle) and TDP-43 + wild-type PDI or TDP-43 + SNO-PDI (open square). The orange and the red lines are drawn through data points indicating the absence of LLPS, whereas the cyan and the blue lines are drawn through data points in which robust LLPS of TDP-43 occurs. The concentration of protein at which these two lines intersect is an estimation of the saturation concentration. ( K and L ) Saturation concentration of TDP-43 (K) and turbidity of TDP-43 condensates (L) (open red circles shown in scatter plots) were expressed as the mean ± SD (with error bars) of values obtained in three biologically independent experiments. TDP-43 + wild-type PDI, P = 0.000002 (K) and 0.00012 (L); TDP-43 + dnPDI, P = 0.0545 or 0.00000068 (K) and 1.0 or 0.00012 (L); and TDP-43 + SNO-PDI, P = 0.0299 or 0.0000000092 (K) and 0.0161 or 0.0029 (L). Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 relative to control (the saturation concentration for TDP-43 or TDP-43 + wild-type PDI). n.s., no significance.

Article Snippet: The following antibodies were used: rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Co-Immunoprecipitation Assay, Transfection, Binding Assay, Western Blot, Construct, Bimolecular Fluorescence Complementation Assay, Expressing, Cell Culture, Staining, Fluorescence, In Vitro, Labeling, Purification, Incubation, Control, Recombinant, Concentration Assay

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Co-IP assay to verify the interaction of endogenous TDP-43 with endogenous G3BP1 in N2a cells transiently expressing empty vector pcDNA3.1, transiently expressing pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h, transiently expressing wild-type PDI treated with 1 mM H 2 O 2 for 1 h, and transiently expressing dnPDI also treated with 1 mM H 2 O 2 for 1 h. Anti-TDP-43 antibody-binding beads were used for co-IP experiments and then detected by western blotting using anti-G3BP1, anti-TDP-43, anti-HA, and anti-β-actin antibodies. ( B ) The normalized amount of immunoprecipitated G3BP1 in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00031; and dnPDI + H 2 O 2 , P = 0.270 or 0.00073. Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; and *** P < 0.001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. (C ) Immunofluorescence imaging of N2a cells transiently expressing both EGFP-TDP-43 and pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibodies against PDI (red) and G3BP1 (magenta) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in (C). White arrows indicate colocalization of TDP-43 and endogenous G3BP1 in stress granules. Scale bar, 10 μm.

Article Snippet: The following antibodies were used: rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Binding Assay, Western Blot, Immunoprecipitation, Control, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Western blot for phosphorylated endogenous TDP-43 (pTDP-43) in N2a cells transiently expressing empty vector pcDNA3.1, transiently expressing pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h, transiently expressing wild-type PDI treated with 1 mM H 2 O 2 for 1 h, and transiently expressing dnPDI also treated with 1 mM H 2 O 2 for 1 h. The cell lysates from the aforementioned cells were probed by anti-TDP-43, anti-pTDP-43 (pSer409/410), anti-HA, and anti-β-actin antibodies. ( B ) The normalized amount of pTDP-43 in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00083; and dnPDI + H 2 O 2 , P = 0.211 or 0.00026. ( C ) Immunofluorescence imaging of N2a cells transiently expressing both EGFP-TDP-43 and pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI also treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibodies against PDI (red) and pTDP-43 (magenta) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in ( C ). Scale bar, 10 μm. ( D ) Western blot for endogenous TDP-43 in the sarkosyl-insoluble pellets (including insoluble full-length TDP-43 and insoluble C-terminal TDP-43 fragment of 35 kDa) and the corresponding cell lysates from the same N2a cells as in (A). β-actin served as the protein loading control. ( E ) The normalized amount of TDP-43 aggregates in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.000091; and dnPDI + H 2 O 2 , P = 0.0109 or 0.00026. Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. ( F to I ) Immunogold electron microscopy of TDP-43 aggregates purified from the same N2a cells transiently expressing pcDNA3.1 (F and G), wild-type PDI (H), or dnPDI (I) treated without (F) or with (G−I) 1 mM H 2 O 2 as in (A), and labeled by gold particles conjugated with anti-TDP-43 antibody. Scale bar, 20 nm.

Article Snippet: The following antibodies were used: rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Western Blot, Expressing, Plasmid Preparation, Immunofluorescence, Imaging, Control, Staining, Electron Microscopy, Purification, Labeling

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Western blot for phosphorylated endogenous TDP-43 (pTDP-43) in HEK-293T cells transiently expressing empty vector pcDNA3.1, transiently expressing pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h, transiently expressing wild-type PDI treated with 1 mM H 2 O 2 for 1 h, and transiently expressing dnPDI also treated with 1 mM H 2 O 2 for 1 h. The cell lysates from the aforementioned cells were probed by anti-TDP-43, anti-pTDP-43, anti-HA, and anti-β-actin antibodies. ( B ) The normalized amount of pTDP-43 in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00027; and dnPDI + H 2 O 2 , P = 0.977 or 0.00095. Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; and *** P < 0.001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. ( C ) Immunofluorescence imaging of HEK-293T cells transiently expressing both EGFP-TDP-43 and pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI also treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibodies against PDI (red) and pTDP-43 (magenta) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in (C). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii

doi: 10.1101/562587

Figure Lengend Snippet: A, B) Microscopic images of T. gondii parasites showing localization of an HA tagged TgTPx1/2 (red) with a mitochondrial marker SPTP-SOD2-GFP (green) and an apicoplast marker ACP (green). C) A Western blot analysis for total cell lysate of parasites stably expressing TgTPx1/2-HA using anti-HA antibodies. Full-length Western blot is represented in Supplementary Fig. S3. D, E) Fluorescence images of parasites stably expressing TgTPx1/2N 1-50 -EGFP (green) with a mitochondrial marker SPTP-SOD2-DsRed (red) and apicoplast maker, ACP (red). Scale bar, 2µm. Numbers (in black) at the top right corner of the DIC+Merge panel indicates the number of parasites inside the vacuole.

Article Snippet: The proteins were transferred to PVDF membrane, blocked for an hour with 5% BSA-Tris Buffered Saline (TBS) and probed with primary antibodies anti-HA (C29F4) Rabbit monoclonal antibody (mAb) (Cell Signaling Technology®) or mouse raised anti-GFP antibodies (Roche) for 2 hours.

Techniques: Marker, Western Blot, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii

doi: 10.1101/562587

Figure Lengend Snippet: A) SignalP 3.0-HMM and MitoProt scores for the wildtype TgTPx1/2 and the mutants TgTPx1/2(R24A) and TgTPx1/2(L17A,L27A). Immunofluorescence images of the parasites expressing B, C) TgTPx1/2(R24A) (red/green) and D, E) TgTPx1/2(L17A,L27A) (green/red). Here ACP is used to label the apicoplast (green) while Mitotracker Red (red) is used for labelling the mitochondrion of T. gondii . Scale bar, 2µm. Numbers (in black) at the top left corner of the DIC panel indicates the number of parasites inside the vacuole. F) A Western blot analysis for the whole parasite lysates of wildtype TgTPx1/2, TgTPx1/2(R24A) and TgTPx1/2(L17A,L27A) using anti-HA antibodies. Full-length Western blot is represented in Supplementary Fig. S3.

Article Snippet: The proteins were transferred to PVDF membrane, blocked for an hour with 5% BSA-Tris Buffered Saline (TBS) and probed with primary antibodies anti-HA (C29F4) Rabbit monoclonal antibody (mAb) (Cell Signaling Technology®) or mouse raised anti-GFP antibodies (Roche) for 2 hours.

Techniques: Immunofluorescence, Expressing, Western Blot