EMBO/soda-nel huggingface dataset:
Nitric oxide‐induced mitochondrial fission is regulated by dynamin‐related GTPases in neurons (2006)
Figure 7

Mitochondrial fission is required for neuronal cell death and mediated by Drp1 and Mfn1. Neurons were co‐transfected with Mito‐DsRed2 plus the plasmids encoding the indicated protein(s). (A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25–35 versus Aβ35–25 (10 μM, 6 h), as indicated. Scale bar, 20 μm. (B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining. (C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments). (D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4). (E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3). (F) Mitochondrial fission by Aβ35–25 or Aβ25–35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25–35 treated neurons; n=3).
Rotational motion and rheotaxis of human sperm do not require 
functional CatSper channels and transmembrane Ca2+ signaling (2019)
Figure 1

Figure 1: Characterization of CATSPER2-deficient human sperm.

(A) Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- sperm

(B) Representative monovalent CatSper currents in CATSPER2-/- sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black) and corresponding current-voltage relationship (right). The membrane voltage was stepped from -100 mV to +100 mV in increments of 10 mV from a holding potential of -80 mV.

(C) Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2-/- sperm (color code: panel B) and sperm from healthy donors (black).

(D, E) Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm.

(F) 3D-STORM image in xy-projection of sperm from a healthy donor labelled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.

(G) 3D-STORM images in xy-projection of CATSPER2-/- sperm (left) labelled with the anti-CatSper 3 (upper panel) and anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.
Rotational motion and rheotaxis of human sperm do not require 
functional CatSper channels and transmembrane Ca2+ signaling (2019)
Figure 5

Figure 5: Rolling behavior and rheotaxis of mouse sperm.

(A) Left: Bright-field image series of a freely swimming wild-type sperm cell at t = 0, 151, and 303 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming wild-type sperm.

(B) Left: Bright-field image series of a freely swimming Catsper1-/- sperm cell at t = 0, 151, and 294 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming Catsper1-/- sperm.

(C) Representative trajectories of wild-type sperm in the absence of a fluid flow. The starting point of each trajectory was centered to the origin of a coordinate system, represented by the intersection of the dotted lines in the center of the circle. Each color represents one trajectory.

(D) Spider-web plot of the mean (± SD) angular frequencies (binning: 315°-45°, 45°-135°, 135°-225°, and 225°-315°; 127 sperm; 3 experiments) in the absence of a fluid flow.

(E) Representative trajectories of wild-type sperm in the presence of a fluid flow.

(F) Spider-web plot of the mean (± SD) angular frequencies (175 sperm; 3 experiments) of wild-type sperm in the presence of a fluid flow. The red arrow indicates the flow direction.

(G) Representative trajectories of Catsper1-/- sperm in the absence of a fluid flow. The starting point of each trajectory was centered to the origin of a coordinate system, represented by the intersection of the dotted lines in the center of the circle. Trajectories are magnified by a factor of 2.05 with respect to the plots C and E to compensate for the reduced swimming speed of the Catsper1-/- sperm.

(H) Spider-web plot of the mean (± SD) angular frequencies of Catsper1-/- sperm (297 sperm; 4 experiments) in the absence of a fluid flow.

(I) Representative trajectories of Catsper1-/- sperm in the presence of a fluid flow; Trajectories are magnified by a factor of 2.05 with respect to the plots C and E to compensate for the reduced swimming speed of the Catsper1-/- sperm, two trajectories were truncated (indicated by two parallel lines).

(J) Spider-web plot of the mean (± SD) angular frequencies (261 sperm; 4 experiments) of Catsper1-/- sperm in the presence of a fluid flow. The red arrow indicates the flow direction.
Protective anti-prion antibodies in human immunoglobulin repertoires (2020)
Figure 2

Figure 2: Binding specificities of retrieved anti-PrP Fabs by ELISA.

(A) ELISA titration curves (OD at 450nm) of Fab3 (blue; left panel) and Fab71 (blue; right panel) against mouse recPrP23‑231 compared to their best affinity-matured versions, Fab83 and Fab101 (red; EC50 values in Dataset EV1). Curves were fitted by non-linear least-squares regression analysis for statistical analysis: log(agonist) vs. response.

(B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab).

Data information: ELISA data were performed in duplicates. Data represent the mean ± sem.
Protective anti-prion antibodies in human immunoglobulin repertoires (2020)
Figure 3

Figure 3: Fabs targeting the CC1 and OR of PrPC prevent prion-induced neurotoxicity in COCS.

(A) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 (CC123‑50), Fab71 (OR51‑91) or Fab1 (GD) for 45 days. Scale bar: 500 μm.

(B) Same as (A), but in the presence of the affinity-maturated antibodies.

(C) NeuN immunofluorescence coverage of prion-infected slices cultured in the presence of the Fabs. Antibodies targeting the CC123‑50 and the OR51‑91 afforded protection against neurodegeneration. NBH: non-infectious brain homogenate. Each dot represents a cerebellar slice for all treatment groups.

Data information: Data are indicated as the mean ± sem. Two-way ANOVA followed by Bonferroni's post-hoc test; ** p<0.01; *** p<0.001; **** p<0.0001.

(D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.

Data information: Data are indicated as mean ± sem. One-way ANOVA followed by Bonferroni's post-hoc test was used for statistical analysis: ** p<0.01, n.s.: not significant.
Protective anti-prion antibodies in human immunoglobulin repertoires (2020)
Figure 4

Figure 4: Identification of anti-PrP Fab71 analogous antibodies in human antibody repertoires.

(A) Fab71-similar HCDR3 sequences in three different NGS datasets (DW: dataset for VH:VL; BB and DK datasets for VH) of human antibody repertoires from healthy donors. Pie chart and stacked bar plot (middle) indicate the occurrence of the identified Fab71 similar HCDR3s: 555 out of 629 identified sequences were only found in one donor, while 74 sequences were shared between donors and occurred in 2 up to 6 donors. Stacked bar plots (right) report the number of sequences differing to Fab71 HCDR3 by 3, 2 or 1 residues. In total, 13 sequences differed by only one residue. Among them, 4 sequences were shared between different donors.

(B) Sequence alignment of Fab71 VH3-30 with HCDR3 regions from the different databases that differ from Fab71 by ≤ 3 residues (upper panel). Amino acids differing from Fab71 are highlighted by orange boxes. Lower panel: sequence alignment of Fab71 light chain VK3-15 with DK_VL2-14 that is naturally paired with DK_VH3-07.

(C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.
Protective anti-prion antibodies in human immunoglobulin repertoires (2020)
Figure 5

Figure 5: Identification of anti-PrP autoantibodies in an unselected hospital patient cohort.

(A) Schematic workflow for the HTS of patient plasma samples for anti-PrP autoantibodies.

(B) Age pyramid, separated for females and males, of the 37,894 unselected hospital patients tested in the HTS.

(C) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum).

(D) Frequency distribution of hits based on age.

(E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity.

(F) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction.

(G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2).

(H) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.

Data information: ELISA data were performed in duplicates. Data represent the mean ± sem.

Expanded View figure legends
ArfGAP1 inhibits mTORC1 lysosomal localization and activation (2021)
Figure 1

Figure 1. ArfGAP1 and mTORC1 interact in cells.

(A) Human embryonic kidney 293A (HEK293A) cells were transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 as a control for 24 hours. Cell lysates were immunoprecipitated with anti-HA beads. Immunoprecipitates (IP) or whole cell lysate (WCL) samples were probed for HA and the mTORC1 components (mTOR, Raptor and mLST8). Vinculin was probed as a control.

(B) Wildtype (Ctrl) or Raptor endogenously GFP-tagged (Raptor-GFP) HAP1 cells were immunoprecipitated with GFP antibody and protein A/G beads. IP or WCL samples were probed for GFP and ArfGAP1.

(C) Flag-tagged Raptor was transfected with HA-tagged ArfGAP1 or HA-tagged RFP1 in wildtype (WT) or RagA/B knockout (KO) HEK293A cells for 24 hours. Cell lysates were immunoprecipitated (IP) with anti-Flag beads. IP or WCL samples were probed for HA-tagged ArfGAP1 or HA-tagged RFP1, Flag-tagged Raptor, RagA, RagB, and Vinculin the loading control.

(D) RagA/B KO HEK293A cells (control or stably overexpressing Flag-tagged Raptor) were transfected with HA-tagged ArfGAP1 or empty vector for 24 hours. Cell lysates were immunoprecipitated with anti-Flag or anti-HA beads as indicated. IP or WCL samples were probed for HA-tagged ArfGAP1, Flag-tagged Raptor, and other mTORC1 components mTOR and mLST8.

(E) HEK293A cells were transfected with empty vector (EV) or Flag-tagged Raptor with or without HA-tagged ArfGAP1 for 24 hours. Cell lysates were immunoprecipitated (IP) with anti-Flag beads. IP and WCL samples were probed for Flag-tagged Raptor, HA-tagged ArfGAP1, mTOR, and the phosphorylation of mTOR at Serine 2481.

(F) Schematic of distribution and comparison of domains between human ArfGAP1, ArfGAP2, and ArfGAP3 proteins.

(G) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP2, or HA-tagged RFP1 (control) for 72 hours. Cell lysates were immunoprecipitated (IP) with anti-HA beads. IP or WCL samples were probed for HA and mTORC1 components mTOR, Raptor and mLST8. Vinculin was probed as a control.
ArfGAP1 inhibits mTORC1 lysosomal localization and activation (2021)
Figure 3

Figure 3. Live cell quantification of ArfGAP1-Raptor interaction under different nutrient conditions.

(A) Left- HA-tagged ArfGAP1 was transfected in RagA/B knockout (KO) human embryonic kidney 293A (HEK293A) cells. Immunofluorescence images of HA-tagged ArfGAP1 (green) and mTOR (red) were assessed for co-localization (scale bar, 5 μm). Right- Line-scan analysis was performed. Yellow line (left) on image measures intensity along line. Distance across the yellow line is measured in microns.

(B) Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster resonance energy transfer (FRET) analysis were performed in wildtype (WT, left) or RagA/B KO (middle) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). At least 20 cells were analyzed in each group. Box plots shows the percentage of pixels with ≥ 30% FRET efficiency of co-transfected cells. RFP1 was expressed and analyzed as a negative control. The whiskers of the boxplot represent the minimum and maximum values, while the box covers the data between 25th and 75th percentiles with a line in the middle plotted at the median. Right- Expression level of RagA/B was confirmed Western blot analysis. Actin was used as a control.

(C) Phasor analysis was performed for the FLIM/FRET experiments described in (B) in WT (left) or RagA/B KO (right) HAP1 cells expressing mCherry-tagged ArfGAP1 and endogenously GFP-tagged Raptor. Cells either had amino acids (+AA) or were starved of amino acids (-AA). For each condition, the first panel shows the fluorescence signal of the green channel. The second panel shows the phasor distribution of GFP (black circle), GFP-tagged Raptor (green circle), and GFP-tagged Raptor plus mCherry-tagged ArfGAP1 (red circle). The green line indicates where the GFP phasor would fall when containing autofluorescence. The third panel shows spatial distribution of pixels associated with FRET. Scale bars: 9 μm for WT (left) and 5 μm for RagA/B KO (right) cells, respectively.

(D) Raster Imaging Correlation Spectroscopy (RICS) analyses was performed for WT (left) or RagA/B KO (right) HAP1 cells expressing endogenously GFP-tagged Raptor (green curves) with either mCherry-tagged ArfGAP1 (blue curves) or RFP1 as a control (red curves). Auto-correlation (AC) or cross-correlation (CC) curves are shown for samples in conditions with amino acids (+AA, solid curve) or without amino acids (-AA, dashed curve).
ArfGAP1 inhibits mTORC1 lysosomal localization and activation (2021)
Figure 5

Figure 5. ALPS motifs are crucial for ArfGAP1 to interact with and inhibit mTORC1.

(A) Schematic of domain distribution of wildtype human ArfGAP1 (amino acids 1-406) and the COPI defective mutant of ArfGAP1 (amino acids 1-394). Critical residues of various domains are highlighted.

(B) RagA/B knockout (KO) human embryonic kidney 293A (HEK293A) cells were transfected with HA-tagged wildtype ArfGAP1, the HA-tagged ArfGAP1 R50K mutant, or empty vector (EV, control) for 72 hours. Cell lysates were immunoprecipitated with anti-HA beads. IP or WCL samples were probed for HA and mTORC1 components (mTOR, Raptor and mLST8). Vinculin was probed for as a control.

(C) RagA/B KO HEK293A cells were transfected with HA-tagged wildtype ArfGAP1, HA-tagged ArfGAP1 L207D/V279D, or RFP1 (control) for 24 hours. Cell lysates were analyzed as in (B).

(D) Top - Input of GST, GST-tagged ArfGAP1, and GST-tagged ArfGAP1 L207D/V279D that was us in the in vitro binding assays. Bottom - Flag-tagged mTOR, Flag-tagged Raptor, and Flag-tagged mLST8 were overexpressed in HEK293A cells. Cell lysates were immunoprecipitated (IP) with anti-Flag beads and incubated with either GST (control), GST-tagged ArfGAP1, or GST-ArfGAP1 L207D/V279D. IP or WCL samples were probed for Flag or GST.

(E) RagA/B KO HEK293A cells were transfected with HA-tagged wildtype ArfGAP1, 1-394 truncation of ArfGAP1, or RFP1 as a control for 24 hours. Cell lysates were immunoprecipitated with anti-HA beads. Immunoprecipitates (IP) or whole cell lysate (WCL) samples were probed for HA, AP-2, or β-COP. Actin served as a loading control.

(F) RagA/B KO HEK293A cells were transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP1 1-394, or RFP1 (control) for 24 hours. Cell lysates were immunoprecipitated with anti-HA beads. Cell lysates were analyzed as in (B).

(G) RagA/B KO HEK293A cells were co-transfected with HA-tagged ArfGAP1, HA-tagged ArfGAP1 L207D/V279D, or RFP1 (control) together with Myc-S6K1. Cells were starved of amino acids (-AA) for 2 hours and then 4 mM of Gln or Asn were added for 2 hours. Cell lysate were immunoprecipitated (IP) with anti-Myc beads. IP or WCL samples were probed for HA, Myc, and phosphorylated S6K1 at threonine 389 (pS6K1). Actin served as a loading control.
ArfGAP1 inhibits mTORC1 lysosomal localization and activation (2021)
Figure 6

Figure 6. ArfGAP1 is associated with more favorite clinical outcome of pancreatic cancer patients and suppresses pancreatic cancer growth.

(A) TCGA cohort of pancreatic cancer patients were divided into two groups according to the median level of ARFGAP1 mRNA expression. Overall survival and disease-free survival were compared between these two groups, as shown in Kaplan-Meier curves. Median survivals (in months), log-rank P values, and hazard ratios (HR) with 95% confidence intervals are indicated.

(B) MIA Paca-2 cells were transfected with HA tagged ArfGAP1 or RFP1 (control) for 4 days and then cell numbers were counted. Six replicated wells were counted each group. Columns represent Mean ± SEM. Student's t-test *P = 0.028.

(C) Left- Expression levels of ArfGAP1 and phosphorylation of S6K1 (pS6K1) at threonine 389 (mTORC1 activity) were analyzed by immunoblotting. Vinculin was a loading control. Right- MIA Paca-2 cells stably expressing shRNAs against ArfGAP1 or GFP as a control were treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 24 hours, and then cell diameters were determined using a particle size counter. The curves show the overall distribution of cell size in each group. Mean of average size ± SD (N = 4): shGFP Vehicle 19.55 ± 0.10, shGFP Torin 1 18.76 ± 0.12, shArfGAP1 Vehicle 20.63 ± 0.16, shArfGAP1 Torin 1 19.05 ± 0.09. Student's t-test P values: shGFP Vehicle vs. shGFP Torin 1 (P = 4.891E-05), shGFP Vehicle vs. shArfGAP1 Vehicle (P = 2.546 E-05), shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 2.38 E-06), and shGFP1 Torin 1 vs. shArfGAP1 Torin 1 (P = 0.007).

(D) MIA Paca-2 cells stably expressing short hairpin RNAs (shRNAs) against ArfGAP1 or GFP (control) were plated and treated with mTOR inhibitor Torin 1 (250 nM) or vehicle control for 48 hours prior to cell counting. Columns represent Mean ± SEM (N = 6). Student's t-test shGFP Vehicle vs. shGFP Torin 1 (P = 2.83 E-07), shGFP Vehicle vs. shArfGAP1 Vehicle (*P = 0.025), shGFP Torin 1 vs, shArfGAP1 Torin 1 (P = 0.139), and shArfGAP1 Vehicle vs. shArfGAP1 Torin 1 (P = 1.22 E-07). "ns" stands for "not significant".
Neurofilaments in spinocerebellar ataxia type 3: blood biomarkers at the preataxic and ataxic stage in humans and mice (2020)
Figure 5

Figure 5. Neurofilament plasma levels and brain tissue levels of soluble and aggregated ataxin-3 in the 304Q SCA3 mouse model.

(A-B) Plasma concentrations of NfL (A) and pNfH (B) were measured in the 304Q knock-in SCA3 mouse model. Heterozygous animals become symptomatic at ≈8 months of age.

(C-F) Tissue levels of soluble and aggregated mutant ataxin-3 were measured in cerebellum (C, E) and frontal lobe (D, F). Exploratory analyses in homozygous animals confirmed the ataxin-3 increases observed in heterozygous animals (Fig EV4).

Heterozygous and wildtype animals were compared by two-tailed unpaired t tests, adjusted for unequal variances (*** p<.001, ** p<.01, * p<.05, ns p≥.05, Bonferroni-corrected; see Appendix Table S3 for detailed statistics). Dots show individual measurements, bars indicate mean ± SD (calculated for log-transformed values).
Neurofilaments in spinocerebellar ataxia type 3: blood biomarkers at the preataxic and ataxic stage in humans and mice (2020)
Figure 7

Figure 7. Ataxin-3 immunohistochemistry and histological assessment of Purkinje cells in the 304Q SCA3 mouse model.

(A) We assessed samples of cerebellar cortex, deep cerebellar nuclei, frontal cortex, pons and pyramidal tract by immunohistochemistry with ataxin-3 staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals at 18 months.

(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals.

ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.

Tables

Table 1. Demographic, clinical and genetic characteristics of the human SCA3 cohorts.

Data are reported as median and interquartile range. NA: not applicable.

Expanded View Figure legends