EMBO/soda-nel huggingface dataset:
Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation (2022)
Figure 1

Figure 1. TDP-43 phosphorylation by CK1δ and C-terminal phosphomimetic substitutions reduce TDP-43 condensation in vitro.

A Scheme of sedimentation assay (created in BioRender.com): Phase separation of TDP-43 was induced by TEV protease cleavage of TDP-43-MBP-His6, and condensates were pelleted by centrifugation.

B Sedimentation assay to quantify condensation of unmodified TDP-43 versus in vitro phosphorylated TDP-43 (+CK1δ, +ATP) and controls (CK1δ or ATP only); TDP-43 detected by Western blot (rabbit anti-TDP-43 N-term). Due to incomplete TEV cleavage, some TDP-43-MBP-His6 remains present and co-fractionates with cleaved TDP-43, due to TDP-43 self-self interaction.

C Quantification of band intensities of cleaved TDP-43 shown as means of Supernatant/(Supernatant+Condensate) [S/(S+C)] ratio of three independent experimental replicates (n=3) ± SD. ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt.

D Schematic diagram of TDP-43 and sequence of C-terminal region (aa. 370 - 414) for Wt, phosphomimetic (S-to-D) variants and control (S-to-A) variants. NTD = N-terminal domain; RRM = RNA recognition motif, LCD = low complexity domain with α-helical structure.

E Turbidity measurements (optical density (OD) at 600 nm) to quantify phase separation of the indicated TDP-43 variants at three different concentrations (in Hepes buffer). Values represent means of three independent experimental replicates (n=3) ± SD. *p < 0.0332, **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt, comparing the respective concentration condition (5, 10, 20 µM).

F-I Representative bright field microscopic images of TDP-43 condensates (in Hepes buffer), Bar, 25 µm (F) and quantification of condensate number (G), size (H) and roundness (I). Box plots show the comparison of median and inter-quartile range (upper and lower quartiles) of all fields of view (FOV) from Min to Max (whiskers) of two replicates (≥ 22 FOV per condition). *p < 0.0332, **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt, comparing the respective concentration condition (5, 10, 20 µM).
Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation (2022)
Figure 2

Figure 2. C-terminal phosphomimetic substitutions enhance liquidity of TDP-43 condensates and reduce TDP-43 aggregation in vitro.

A Representative still images of Alexa488-labeled TDP-43 condensates by spinning disc timelapse confocal microscopy. Wt condensates do not fuse, 5D condensates fuse slowly and 12D condensates readily fuse upon contact and relax into spherical droplets. Bar, 5 µm.

B Representative images of FRAP experiments at indicated time-points. Boxes indicate bleached area (half-bleach of condensate). Bar, 5 µm.

C FRAP curves after half-bleach of freshly formed Alexa488-labeled TDP-43 condensates. Values represent means ± SD of three independent experimental replicates (n=3) of ≥ 9 droplets analyzed per condition. ***p < 0.0002 by one-way ANOVA with Tukey´s multiple comparison test for area under the curve (AUC) of individual droplets.

D Confocal images of Alexa488-labeled TDP-43 aggregates formed in an in vitro aggregation assay (with TEV protease cleavage). Bar, 100 µm. Zoom shows magnified view of aggregates at the 24h time point. Bar, 20 µm.

E SDD-AGE followed by TDP-43 Western blot to visualize SDS-resistant oligomers / high molecular weight species of TDP-43-MBP-His6 in an in vitro aggregation assay (without TEV protease cleavage). Asterisks represent monomeric (*), oligomeric (**) and polymeric (***) species.

F Input of TDP-43-MBP-His6 variants used in the SDD-AGE assay, detected by Western blot (anti-TDP-43 N-term).
Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation (2022)
Figure 4

Figure 4. Phosphomimetic substitutions do not alter the rate of TDP-43 nuclear import and do not impair TDP-43 autoregulation, RNA-binding or alternative splicing function.

A Hormone-inducible nuclear import assay, representative still images of GCR2-EGFP2-TDP-43 Wt, 12D and 12A before and during import triggered by addition of dexamethasone. Images were live recorded by spinning disc confocal microscopy. Bar, 20 µm.

B Quantification of the hormone-inducible nuclear import measured during a total time course of 50 min. Values represent the mean fluorescence intensity of GCR2-EGFP2-TDP-43 in the cytoplasm for three independent replicates ± SEM (≥ 42 cells per condition).

C Phosphomimetic 12D TDP-43 is competent in autoregulating TDP-43 expression. SDS-PAGE followed by TDP-43 Western blot showing downregulation of endogenous TDP-43 through autoregulation (60) after 48 h expression of Wt, 12D and 12A variants in HeLa cells. TDP-43 was detected using rabbit anti-TDP-43 C-term antibody (Proteintech), Histone H3 (rabbit anti-Histone H3 antibody, Abcam) was visualized as a loading control. * denotes an unspecific band.

D Electrophoretic mobility shift assays (EMSA) of TDP-43-MBP-His6 variants (Wt, 12D and 12A) in a complex with TDP-43 autoregulatory RNA binding site (60). All TDP-43 variants form TDP-43-RNA complexes equally well.

E Splicing analysis by RT-PCR of known TDP-43 splice targets (SKAR exon 3 and Bim exon 3) in HeLa cells. Silencing of endogenous TDP-43 by siRNA leads to altered splice isoforms of SKAR and Bim (second vs. first lane). These splicing alterations can be rescued by re-expression of TDP-43 Wt, but also 12D or 12A variants, demonstrating that phosphomimetic TDP-43 is fully competent in regulation splicing of these TDP-43 splice targets.
Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation (2022)
Figure 5

Figure 5. Phosphorylation and phosphomimetic substitutions reduce recruitment of TDP-43 into stress-induced membrane-less organelles.

A Scheme of stress granule (SG) recruitment assay in semi-permeabilized cells.

B Reduced SG association of TDP-43 by 12D mutations or in vitro phosphorylation. Bar, 20 µm.

C Quantification of TDP-43-MBP-His6 mean fluorescence intensity in SGs normalized to Wt ± SEM of three independent experimental replicates (n=3) (≥ 10 cells; ≥ 46 SGs each). **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt.

D SG recruitment of TDP-43 variants in intact HeLa cells in absence of endogenous TDP-43. After TDP-43 silencing and expression of myc-TDP-43 Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and SG recruitment of TDP-43 was monitored by TDP-43 and TIA1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP-43 (green) and TIA-1 (magenta). Bar, 25 µm.

E Quantification of TDP-43 in SGs versus cytoplasm ± SD of two independent experimental replicates (n=2) (≥ 62 cells; ≥ 234 SGs each). ***p < 0.0002 by Krustal-Wallis test with Dunn´s multiple comparison test to Wt.

F SG recruitment of different TDP-43-NLSmut variants in intact HeLa cells in the absence of endogenous TDP-43. After TDP-43 silencing and expression of NLSmut Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and SG recruitment of TDP-43 was monitored by TDP-43 and G3BP1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP-43 (green) and G3BP1 (red). Bar, 40 µm.

G Quantification of TDP-43-NLS mutants in SGs versus band around SGs of two independent replicates ± SD. ***p < 0.0002 by Kruskal-Wallis test with Dunn´s multiple comparison test to Wt (≥ 56 cells; ≥ 315 SGs per condition).

H Recruitment of TDP-43 into arsenite-induced nuclear bodies (NBs) in HeLa cells. After TDP-43 silencing and expression myc-TDP-43 Wt, 12D and 12A, NBs were induced by sodium arsenite treatment and NB recruitment of TDP-43 was monitored by TDP-43 immunostaining. Bar, 20 µm.

I Percentage of cells with TDP-43 in NBs ± SD of three independent experimental replicates (n=3). *p < 0.0332 by Kruskal-Wallis test with Dunn´s multiple comparison test to Wt.

J Intensity profiles (right) of TDP-43 Wt, 12D and 12A variants (green) along white lines (left). Bar, 10 µm.
Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation (2022)
Figure 6

Figure 6. Phosphomimetic substitutions enhance TDP-43 solubility in HeLa cells and primary neurons.

A Biochemical fractionation into RIPA-soluble (S) and RIPA-insoluble (I) fractions to analyze solubility of the different myc-TDP-43 variants (Wt, 12D and 12A) expressed in HeLa cells for 48 h. TDP-43 was detected by TDP-43 Western blot (upper blot, rabbit anti-TDP-43 C-term, Proteintech) and Myc Western blot (lower blot, mouse anti-Myc 9E10).

B Quantification of myc-TDP-43 variants (Wt, 12D and 12A) in (S) versus (I) fractions extracted from TDP-43 Western blots of 4 independent replicates ± SD, plotted as S/(S+I). *p < 0.0332 by one way ANOVA with Dunnett´s multiple comparison test to Wt.

C RIPA-insoluble material of the indicated EGFP-tagged TDP-43 variants (± NLS mutation) expressed in primary cortical neurons analyzed by filter-trap assay.

D Primary hippocampal neurons expressing EGFP-TDP-43 Wt, 12D or 12A with additional NLS mutation. Bar, 80 µm. Right: Zoomed images of white squares (TDP-43 signal). Bar, 10 µm.

E SG recruitment of EGFP-TDP-43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1h heat shock at 42°C. SGs and TDP-43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP-TDP-43 shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.

Expanded View Figure legends
Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins (2016)
Figure 2

Figure 2. Cellular validation of ATG8 sensors

A. Schematic illustration of annexin A4 based co-translocation assay of potential mATG8 binding peptides and their target mATG8 to membranes upon calcium influx induced by ionomycin treatment.

B, C, E. Annexin A4 based co-translocation assays of peptide binders and EGFP-mATG8s. In comparison to single peptides (B), triplication of peptides (C) or introduction of negative charges adjacent to the binding motif (E) increases the co-translocation extent of the peptide-sensors with their target mATG8. Control assays with non-target mATG8 are shown in expanded view figure 2.

D. Biotinylated octameric peptides representing a single amino acid walk-through along the complete 16mer peptide AS_67 and AS_M67 are immobilized on streptavidin. Binding of GST-LC3C fusion-protein to the immobilized peptides is detected by a GST specific antibody (HRP-conjugate) and shown as absorbance at 450 nm. AU=arbitrary unit

F. Translocation efficiency of the unmodified peptide AS_67, triplicate AS3_67 and charge optimized peptide AS_M67 is classified as % of cells that show no translocation ‘–‘ to very good ‘++++’ translocation with EGFP-LC3C; n = total number of analyzed cells. Data are presented as mean of three biological replicates ±SD.
Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins (2016)
Figure 3

Figure 3. ATG8 sensors are recruited to autophagosomes upon autophagy induction

A, B. The recruitment of the mCherry fused p62 derived sensor AS3_p62 to the autophagosomes positive for overexpressed EGFP-LC3B (A) or endogenous LC3B (B) visualized by immunofluorescence upon autophagy induction for 3h by KU + Baf treatment.

C. Recruitment of AS3_p62 in Atg5+/+ or Atg5-/- MEFs to endogenous Lc3b.

D. Lack of recruitment of the p62-derived sensor in the absence of the hydrophobic core residues of the LIR motif (ΔLIR) to EGFP-LC3B positive autophagosomes.

E, F. ATG8 sensors are recruited to autophagosomes decorated with respective target mATG8 proteins. Experiments are performed in HeLa cells (A, B, D, E and F) and MEFs (C), transiently (A-C, D, F) or stably (D) expressing indicated ATG8 sensors. Peptide sequences of the tested mATG8 sensors are listed in expanded view table 1.

G. Existing sensors can be further improved by triple repeats of the engineered peptide (mCh-AS3_X), by engineering flanking charges (mCh_AS_MX), by introduction of an oligomerization domain (mCh-PB1-AS_X) or by increasing the membrane association (FYVE-mCh-AS_X).
Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins (2016)
Figure 4

Figure 4 – Autophagy flux is unaffected by sensors

A-C. Analysis of autophagy flux in stable inducible HeLa cell lines expressing mCherry-fusion proteins of indicated ATG8 sensor upon doxycycline (DOX) induction: Autophagy flux is monitored in basal conditions (A), upon starvation (EBSS) (B), or after autophagy induction by Torin I (C) indirectly via lipidation of LC3B and turnover of the autophagy receptor p62.

D. FACS based mitophagy flux analysis performed in HeLa cell line stably expressing HA-Parkin, mt-mKeima, and a GFP fusion of the indicated sensor. Cells were treated with OAQ (oligomycin, antimycin, Q-VD) for indicated times and analyzed by FACS for lysosomal-positive mt-mKeima (561nm). The average of three independent experiments is presented. Representative FACS data from experiments (GFP control and AS3_67) are shown in the lower panels. Error bars indicate standard deviation. Significance calculation: 2-way anova of one-way anova. n.s.= not significant. *** indicates p-value of 0.0001.
Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins (2016)
Figure 5

Figure 5. Performance of ATG8 sensors can be increased by introduction of the oligomerization domain PB1

A. LC3C specific sensor mCh-AS3_67 is recruited to Salmonella (SFH2) escaping SCV. Salmonella SFH2 strain is carrying a GFP-reporter gene activated by availability of glucose-6-phosphate in the host’s cytosol.

B. Salmonella with prolonged cytosolic residence and strong activation of the GFP-reporter gene (green arrow) are not recognized by the LC3C specific sensor mCh-AS3_67.

C. A subpopulation of hyper proliferative Salmonella in the cytosol is coated with endogenous LC3C. In contrast, endogenous LC3B is enriched in spots in close proximity to the cytosolic bacteria.

D. Sensitivity of LC3C specific sensor mCh-AS3_67 is increased upon fusion of membrane-tethering FYVE domain or PB1 oligomerization domain.

E. Introduction of the PB1 domain to mCh-AS3_p62 increases its performance in recognizing endogenous LC3B.

F. High-performance sensor mCh-PB1-AS3_67 was transiently expressed in Salmonella infected HeLa cells to monitor xenophagy events.

All experiments performed in HeLa cells, except for (E), which was performed in U2OS cells.
Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins (2016)
Figure 6

Figure 6. LC3C specific ATG8 sensor monitoring mitophagy

A,B. LC3C specific sensor mCh-PB1-AS3_67 is recruited to a subset of mitochondria undergoing mitophagy. Mitochondria were labeled with MitoTracker Deep Red (ThermoFisher). U2OS cells were transiently co-transfected for indicated sensor and mATG8 proteins. Mitophagy was induced by CCCP treatment over three hours. Lysosomal degradation of mitochondria undergoing mitophagy was blocked by the addition of BafilomycinA1 (Baf).

C. Upon depolarization, endogenous LC3C is localized to mitochondria labeled with mt-mCherry in U2OS cells. An example of rare events with nearly complete overlap of signals for endogenous LC3C and mitochondria (upper panel) and a representative image (lower panel) is given.

D. Overlap of LC3C specific sensor signal with endogenous LC3C and endogenous LAMP1 in U2OS cells induced for mitophagy.
Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron-enriched genes (2021)
Figure 1

Figure 1. Setd1b is required for hippocampus-dependent memory.

A. qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected (CA: Control, n = 6; cKO, n = 6. DG: Control, n = 6; cKO, n = 6. Cortex: Control, n = 7; cKO, n = 7. Cerebellum: Control, n = 4; cKO, n = 4). * p-value < 0.05 (Student t-test).

B. Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice (Control, n = 4; cKO, n = 4). ** p-value < 0.01 (Student t-test).

C. Immunohistochemical staining (upper level) for marker proteins of neuronal integrity and quantification (lower panel) show no difference in control and Setd1b cKO mice (NeuN: Control, n = 6; cKO, n = 6; Student t-test p-value = 0.57. MAP2: Control, n = 4; cKO, n = 4; Student t-test p-value = 0.72. Iba1: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.8. Gfap: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.09.). Scale bar: 100 µm.

D. Average speed (left panel) and percent time spent in the center (right panel) during exposure to the open field test was similar in control and Setd1b cKO mice (Average speed: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.075. Time spent in center: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.96).

E. Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y-maze test (Control, n = 15; cKO, n = 15; Student t-test p-value = 0.3). F. Depressive-like behavior was measured in the Porsolt forced swim test. There was no significant difference in the % time spent immobile between groups (Control, n = 8; cKO, n = 8; Student t-test p-value = 0.5).

G. Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice (Control: n = 15, cKO: n = 15. Repeated measures ANOVA, genotype effect: F (1,28) = 82.34, **** p-value < 0.0001).

H. Plots showing the specific search strategies during water maze training. Note the failure of Setd1b cKO mice to adopt hippocampus-dependent search strategies. I. The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice (Student t-test: *** p-value < 0.001).

Data information: Bar graphs indicate mean, Error bars indicate +/- S.E.M, "n" indicates biological replicates.
Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron-enriched genes (2021)
Figure 6

Fig. 6. Comparative analysis of learning and single nucleus gene expression of Kmt2a, Kmt2b and Setd1b.

A. Comparison of the escape latency in the 3 different KMT cKO mice during 10 days of training. Although we applied the same experimental setting, the experiments were performed at different time points ((Kerimoglu et al., 2013) (Kerimoglu et al., 2017), and this study). Thus, for comparison we normalized the data to the corresponding control group. In this plot an increase in the fold change of the normalized escape latency depicts the difference to the corresponding control group. Hence, a higher fold change of the normalized escape latency indicates a greater difference to the corresponding control and thus more severe learning impairment. Setd1b cKO (n = 14) vs Kmt2a cKO (n = 13): Repeated measures ANOVA, genotype effect: F (1,25) = 16.83, *** p-value < 0.001. Setd1b cKO (n = 14) vs Kmt2b cKO (n = 22): Repeated measures ANOVA, genotype effect: F (1,34) = 70.66, **** p-value < 0.0001.

B. UMAP plot showing the data from 15661 nuclei. Upper panel shows the different clusters and their corresponding numbers. The lower panel shows the same UMAP plot colored for different groups of cells that are further analyzed in (C) and (D).

C. Violin plots showing the expression of the 6 KMTs as well as marker genes for the different hippocampal cell types according the UMAP shown in lower panel B. Note that Kmt2a and Kmt2c are the highest expressed KMTs across cell types, while the other KMTs shown low to moderate expression.

D. Violin plots showing the expression of the six KMTs and corresponding marker genes within the different clusters representing inhibitory and excitatory neurons. Note that there is no obvious difference of KMT expression between cell types. In agreement with the data shown in panel C, Kmt2a and Kmt2c exhibit the highest expression levels.

E. Bar charts showing the total number of reads/cell that are positive for the corresponding KMT.

F. Left panel shows representative images of RNAscope performed for Kmt2a, Kmt2b and Setd1b. Right panel shows a bar chart quantifying the dots/cell (n = 1500 cells from 2 mice) indicative of the corresponding expression level. Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b (*** p-value < 0,0001, Student t-test).

Data information: Bar graphs indicate mean, Error bars indicate +/- S.E.M. "n" indicates biological replicates.

Expanded View Figure Legends
Rational design of West Nile virus vaccine through large replacement of 3' UTR with internal poly(A) (2021)
Figure 3

Fig. 3. WNV-poly(A) protects C57BL/6 mice from WT WNV challenge.

A Experimental scheme. Groups of four to six-week-old C57BL/6 mice (n=5 per group) were immunized i.p. with 104-107 PFU of WNV-poly(A), PBS (negative control) or mock (untreated). On day 30 post-immunization, mice were challenged i.p. with 3× 107 PFU of WT WNV

B-C Total anti-WNV IgG antibody (B) and neutralizing antibody titers (C) in mouse serum at the indicated days post immunization. (For B and C, the values represent mean ± sd in each group and the horizontal dotted line represents the limit of detection)

D-E Survival (D) and body-weight changes were monitored daily after challenge. (The values represent mean ± sd in each group) (E). F Viremia was quantified by plaque assay on day 2 post-challenge. (The values represent mean ± sd in each group, each symbol represents a mouse)

G-H T-cell responses in vaccinated mice were measured through ELISpot assay. Two groups of C57BL/6 mice (n = 4 / group) were i.p. inoculated with 107 of WNV-poly(A) or equal volume of PBS. ELISpot assay specific for IFN-γ in splenocytes was performed at 7 days post-immunization. Representative images were shown in (G) and the numbers of antibody secreting cells (ASCs) were counted (The values represent mean ± sd in each group, each symbol represents a mouse) (H).

Data information: For statistical analysis, one-way ANOVA analysis and two-way ANOVA analysis were used for B, C, F and E respectively, Log-rank test was performed for D and Mann-Whitney test was used for H. *p<0.5, **p<0.01, *** p<0.001,****p< 0.0001, ns represents not significant
Rational design of West Nile virus vaccine through large replacement of 3' UTR with internal poly(A) (2021)
Figure 4

Fig. 4. Type I IFN signaling pathway is required for WNV-poly(A) attenuation.

A-B Sensitivity of WT WNV and WNV-poly(A) viruses to interferon α (IFN-α) treatment in Vero cells. Vero cells were infected with either WT WNV or WNV-poly(A) virus at an MOI of 10 and treated with different concentrations of IFN-α. The supernatants were harvested at 48 hpi and viral RNA copy numbers were measured by real-time RT-PCR.

C Comparison of growth kinetics between WT WNV and WNV-poly(A) viruses in MEF cells. MEF cells were infected with either WT WNV or WNV-poly(A) virus at an MOI of 0.01. Values indicate mean ± sd from two replicates of one representative experiment.

D Survival rate of IFNAR-/- mice infected with different dosages of WNV-poly(A) (n=5).

E Cell morphology changes of MEF cells infected with WT WNV and WNV-poly(A).

Data information: Three independent assays were performed for above experiments with similar results and data of one representative data was shown. Scale bars represent 50 μm.