EMBO/soda-nel huggingface dataset:
The TOR-dependent phosphoproteome and regulation of cellular protein synthesis (2021)
Figure 4

Figure 4 - The effects of TOR inhibition on S6K phosphorylation and rates of protein synthesis

Kinetic analyses on the identified phosphosites for the S6 kinase homologues in fission yeast, and the changes in rates of protein synthesis in the S6 kinase deletion mutant relative to wild type upon TOR inhibition.

A Relative protein expression levels of the 3 S6 kinase homologues, Psk1, Sck1 and Sck2, plotted against time of Torin1 addition. Respective proteins are represented by the different coloured lines as detailed in the legend.

B Relative phosphorylation level of the T632 phosphosite identified on Sck1 in the 40-minute time course study. Phosphorylation levels of both wild type (green) and tor1∆ (yellow) are presented relative to the value for wild type cells at T = 0 minutes. Grey dashed line indicates 2-fold threshold.

C Relative phosphorylation of the T16 phosphosite identified on Sck2 in the 85-minute time course study (background adjusted detailed in Methods). Grey dashed line indicates 2-fold threshold.

D Changes in rates of protein synthesis upon Torin1 treatment (5 µM) measured for wild type and the S6 kinase deletion mutant (S6K 3x∆) plotted over time. Non-linear regression exponential decay lines were fitted using the curve fitting function on Prism9. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.
The TOR-dependent phosphoproteome and regulation of cellular protein synthesis (2021)
Figure 5

Figure 5 - Examining potential essential translation-related targets and the functional importance of phosphorylation on rates of protein synthesis upon TOR kinase inhibition

Phosphorylation kinetics of common phosphosites identified for 3 essential translation-related proteins, and the effects of the tif471_18A phosphosite mutant on rates of protein synthesis upon Torin1 treatment.

A, B Phosphosites identified on (A) Tif212 and (B) Erf1 respectively showing 2-fold phosphorylation change within 20 minutes of Torin1 (5 µM) addition. Both wild type (green) and tor1∆ (yellow) phosphorylation values are relative to the starting phosphorylation levels before Torin1 addition in wild type cells (T=0 minutes). Grey dashed lines indicate 2-fold threshold.

C, D Relative phosphorylation levels of the 10 common phosphosites of Tif471 plotted against time after Torin1 treatment. Phosphorylation profiles are grouped based on phosphorylation kinetics, where 6 sites showed more than 1.5-fold increase by 10 minutes (C), and the remaining 4 exceeded 2-fold by 30 minutes (D). Grey dashed lines indicate 2-fold threshold.

E, F Changes in rates of protein synthesis of the tif471_18A mutant compared to the wild type control (tif471+) upon Torin1 (5 µM) treatment. Residual rates of protein synthesis after 60 minutes of Torin1 treatment was 17% for the tif471_18A phosphomutant and 4.6% for the tif471+ control relative to starting levels in the respective strains. The two graphs represent the untransformed (E) and log2 transformed (F) rates respectively. Error bars represent the standard deviation (SD) of data aggregated from 3 independent experiments.
Microglial TNF alpha; orchestrates protein phosphorylation in mouse cortical neurons during the sleep period and controls homeostatic sleep. (2022)
Figure 3

Figure 3 - Kinases involved in microglial TNFα phosphomodulation during the light period.

(A) Phosphomodulation of phosphatases and kinases by microglial TNFα. Graphs show percentage of phospho-phosphatases and phosphokinases with at least one changing phosphosite in micTNFα-KO vs. CTL comparison (phosphosites with an adjusted p-value ≤ 0.05 and unique phosphosites) at ZT18 and ZT6.

(B, C) Density of phosphosites on (B) phosphatases and (C) kinases modulated by microglial TNFα at ZT18 and ZT6. Graphs show the number of significantly changed phosphosites in micTNFα-KO vs CTL comparison per protein weight. Each dot is one phosphoprotein. (B) Phosphatases are shown in grey. Regulatory subunits of phosphoprotein phosphatases (reg sub PPP) in blue. (C) Kinases are color-coded according to the major families: calcium/calmodulin regulated kinases (CAMK, red); CDK, MAPK, GSK3 and CLK kinases families (GMGC, blue); protein kinase A, G and C families (AGC, dark green); kinases belonging to other families (others, grey).

(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. Immunoblots at ZT18 in fig S5C. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.

(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).

(F, G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. Immunoblots at ZT18 in fig S5D. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. (F, G) n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) and **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 1

Analysis of autophagosomal maturation in HCV-infected cells.

(A) Western-blot analysis of HCV-infected cells at various time-points post-infection (p.i.) (m.o.i. = 1). Numbers under Rubicon and UVRAG panels indicate their protein levels at different time points relative to their levels in mock-infected cells (i.e., 0 hour p.i.). (B) Fluorescence imaging of HCV infected cells. Stable Huh7.5 cells that expressed mRFP-GFP-LC3 were infected with HCV and fixed at the time points indicated for the analysis of RFP and GFP puncta. Nuclei were stained with DAPI. The boxed areas were enlarged and shown to the right. (C) Upper panel, levels of RFP and GFP puncta after HCV infection relative to those in mock-infected cells, which were arbitrarily defined as 1. Lower panel, percentages of RFP puncta that were positive for GFP (i.e., Yellow/Red ratio). The results represent the average of >50 cells that were analyzed. (D) Fluorescence imaging of RFP, GFP and LAMP1 in HCV-infected cells expressing mRFP-GFP-LC3. The lysosomal marker LAMP1 was stained in blue color. DAPI was not used to stain the nuclei. The colocalization of RFP and GFP will generate the yellow color, and the colocalization of RFP with LAMP without GFP will generate the purple color. The boxed areas were enlarged and shown to the right.
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 2

Suppression of Rubicon expression enhanced the maturation of autophagosomes and inhibited HCV RNA replication.

Huh7.5 cells were transfected with the negative control siRNA (NC) or the Rubicon (Rb) siRNA for 48 hours and then infected with 1 m.o.i. of HCV. (A) Western-blot analysis of cell lysates at different time points after HCV infection. Actin served as the loading control. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. siNC, negative control siRNA; siRb, Rubicon siRNA. (C) Fluorescence imaging of RFP and GFP puncta in cells transfected with the control siRNA (top two panels) or the Rubicon siRNA (bottom two panels). Cells were fixed at 24 and 48 hours after HCV infection for the analysis. Boxed areas in merged images are enlarged and shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells treated with either the control siRNA or the Rubicon siRNA. The results represent the average of >50 cells.
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 3

Over-expression of Rubicon inhibited the maturation of autophagosomes and enhanced HCV RNA replication.

Huh7.5 cells were transfected with the control vector or the Flag-tagged Rubicon expression plasmid for 24 hours followed by infection with HCV. (A) Western-blot analysis of cell lysates at different time points after infection. Mock-infected cells were lysed at 48 hours post-transfection. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. (C) RFP and GFP puncta in cells with the over-expression of Rubicon at different time points after HCV infection. Merged images are shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells transfected with either the control vector or the Rubicon expression plasmid. The results represent the average of >50 cells.
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 4

Over-expression of UVRAG enhanced the maturation of autophagosomes and reduced HCV RNA replication.

Huh7.5 cells were transfected with the control vector or the Flag-tagged UVRAG expression plasmid for 24 hours followed by infection with HCV. (A) Western-blot analysis of cell lysates at different time points after infection. Mock-infected cells were lysed at 48 hours post-transfection. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. (C) RFP and GFP puncta in cells with the over-expression of UVRAG at different time points after HCV infection. Merged images are shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells transfected with either the control vector or the UVRAG expression plasmid. The results represent the average of >50 cells.
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 5

Analysis of the effects of Rubicon and UVRAG on HCV yield.

Cells transfected with the control siRNA (siNC), the Rubicon siRNA (siRb), the Rubicon expression plasmid (pRubicon) or the UVRAG expression plasmid (pUVRAG) were infected with HCV (m.o.i. = 1). The incubation media were harvested at 24 and 48 hours post-infection and used to infect naïve Huh7.5 cells. Cells were either fixed and stained for the HCV core protein for the determination of viral titers (A) or lysed for Western-blot analysis of the HCV core protein (B) two days after infection. The results shown in (A) represent the average of three independent experiments, and the numbers under the core protein panels in (B) indicate the relative core protein levels, with the core protein level of control siRNA transfected cells arbitrarily defined as 1. Actin served as the loading control in (B).
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 6

Effects of Rubicon on autophagosomal maturation and HCV RNA replication in HCV replicon cells.

(A) Increase of Rubicon, UVRAG, p62 and LC3-II in HCV subgenomic RNA replicon cells. Actin served as the loading control. Numbers under Rubicon and UVRAG indicate the protein levels of Rubicon and UVRAG in replicon cells relative to their levels in control Huh7 cells. (B) Effects of nutrient starvation on Rubicon, p62 and LC3 in Huh7 cells. Huh7 cells were nutrient-starved for 2 or 6 hours as indicated and lysed for Western-blot analysis. The replicon cells were used as the control for comparison. (C) Colocalization analysis of GFP-LC3 puncta and lysosomes. Stable Huh7 cells that expressed GFP-LC3 were nutrient-starved for 2 hours and stained with Lysotracker-red for lysosomes. The HCV replicon cells were also stained with Lysotracker-red for comparison. (D) Colocalization efficiency of GFP puncta with Lysotracker-red shown in (C). The results represent the average of >30 cells. (E) Effect of Rubicon knockdown on parental Huh7 cells and HCV replicon cells. Huh7 cells and HCV replicon cells were treated with the control siRNA or the Rubicon siRNA for two days. Cells were then lysed for Western-blot analysis. (F) Relative HCV RNA levels as measured by real-time RT-PCR. HCV replicon cells treated with either the control siRNA or the Rubicon siRNA for two days were lysed for quantification of HCV RNA by real-time RT-PCR. (G) Effect of UVRAG overexpression on HCV replicon cells. HCV replicon cells were transfected with the control vector or flag-UVRAG plasmid for two days. Cells were then lysed for Western-blot analysis. (H) Relative HCV RNA levels as measured by real-time RT-PCR. HCV replicon cells transfected with either the control vector or the flag-UVRAG plasmid for two days were lysed for quantification of HCV RNA by real-time RT-PCR. In (F) and (H), *, p<0.05.
HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. (2015)
Fig 7

Effects of HCV nonstructural proteins on the induction of Rubicon and autophagosomes.

(A) Western-blot analysis of Huh7 cells transfected with the expression plasmids of HA-tagged GST and various HCV nonstructural proteins. Cells were lysed 48 hours after transfection for analysis. The localizations of molecular weight markers are indicated. The asterisk denotes a nonspecific protein band. (B) Analysis of GFP-LC3 puncta in stable Huh7 cells that expressed GFP-LC3. Cells were transfected with various expression plasmids for 48 hours and immunostained with the anti-HA antibody (red color). GFP-LC3 puncta were apparent in cells that expressed HCV NS4B. (C) The average number of GFP-LC3 puncta per cell shown in (B). The results represent the mean of >30 cells.
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 1

Figure 1. RNA binding promotes liquid-like properties of TDP-43 in the dense phase.

A) Condensates formed by purified TDP-43 at 150 mM salt observed by brightfield microscopy at different time points.

B) TDP-43 condensates without RNA control or in the presence of A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) (Supplementary Table 1) at 150 mM NaCl (4 μM TDP-43).

C) RNA binding-deficient TDP-43 mutants generated by site-directed substitutions in RRM1 (F2L) or in both RRM domains (F4L) and substitution of RRM1 and RRM2 with monomeric GFP (N-GFP-C). Phase separation of the mutants without added RNA (control) or in the presence A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) at 150 mM NaCl (4 µM protein).

Data information: Representative images for 3 independent experiments with 2 different protein preparations. Scale bars, 10 μm.
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 2

Figure 2. RNA composed of multiple binding sites potently induces TDP-43 phase separation.

A) Description of RNA oligonucleotide sequences composed of multiple TDP-43 binding sites, A(GU)18, CLIP34 and (AUG12)3, NS, or A(CA)18 Sequence in different colors separates regions that accommodate one TDP-43 molecule, if known. NS, non-specific control RNA.

B) TDP-43 condensates in the presence of the different RNA sequences. TDP-43 and RNA oligonucleotide concentration is 4 μM and 3.9 μM, respectively. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.

C) LLPS measured by turbidity in the same experimental conditions as panel B. Mean and SD from 3 biological replicates with 2 different protein preparations.

D) Lysate from HEK 293 cells expressing a GFP-tagged copy of either wild-type (WT) or F147/149/229/231L (F4L) TDP-43 was added to a mixture of recombinant WT or F4L TDP-43 (5.3 μM, 10% Cy3-labeled protein) and A(GU)6 (22.8 μM), A(CA)18 (7.6 μM), A(GU)18 (7.6 μM), or no RNA control at 250 mM NaCl. Droplets were imaged by brightfield and fluorescence microscopy. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.

E) LLPS measured by turbidity in the same experimental conditions as panel D. Mean and SD of 4 biological replicates using 2 recombinant protein preparations. Analyzed by one-way ANOVA (F(7,24)=69.30, P<0.0001). Sidak's multiple comparisons test was used to compare selected groups. *P<0.05, ****P<0.0001.
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 3

Figure 3. RNA with multiple binding sites modulates TDP-43 phase properties. A) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or increasing A(GU)18 RNA concentration at 250 mM salt. Representative images from 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.

B) Area of droplets plotted at different A(GU)18 concentrations. Mean and SD of >150 droplets from 3 biological replicates using 2 different protein preparations.

C) TDP-43 concentration in the light phase measured as a function of A(GU)18 (blue) or A(CA)18 (red) concentration measured by Bradford protein assay. Mean and SD from >6 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43)

D) Phase separation of TDP-43 (4 μM) at increasing salt concentration without RNA or in the presence of increasing A(GU)18 concentration. Scale bars, 10 μm. Inlay added to top right panel for increased visibility, scale bar 1 μm.

E) Phase diagram derived from panel D, denoting either no droplets or droplet formation for each condition.
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 4

Figure 4. TDP-43 phase separation is regulated by the number of binding sites on RNA.

A) TDP-43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or GU-repeat RNA oligonucleotides of increasing length at 250 mM salt. As indicated, RNA concentration varied to maintain a constant total number of TDP-43 binding sites. Scale bars, 10 μm.

B) The plot shows droplet area quantified as a function of GU-repeat length. Mean and SD of >300 droplets from 3 biological replicates using 3 different protein preparations.

C) TDP-43 concentration in the light phase (Cout) as a function of GU (blue) or CA (red)-repeat length quantified by Bradford protein assay. Mean and SD from >5 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43)
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 5

Figure 5. RNA-induced phase separation requires multivalent interactions mediated by TDP-43 domains.

A) Full-length TDP-43, mutant, and deletion fragments targeting regions that mediate self-assembly. Amino acid residue positions are indicated for each.

B) Phase separation of full-length and TDP-43 variants (4 μM) mixed with A(GU)30 RNA (0.8 μM) or no RNA control at 250 mM NaCl observed by brightfield microscopy. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 6

Figure 6. ALS-linked TDP-43 mutations A321G and M337V impair RNA-driven phase separation.

A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm.

B) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.
Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties (2021)
Figure 7

Figure 7. RNA binding is a key driver of TDP-43 phase separation maintaining liquid properties of the dense state.

A) Binding of TDP-43 to GU-rich RNA promotes liquid properties of TDP-43 condensates, as shown by the fusion of small condensates into larger droplets. This fluidity is impaired by the loss of RNA binding affinity or disease-linked mutations that alter phase separation, such as M337V and A321G, which could explain increased TDP-43 aggregation.

B) RNA molecules that bind multiple TDP-43 molecules (orange lines) provide a scaffold and increase multivalent interactions, including N-and C-terminal domains, creating a meshwork that results in phase separation. In contrast non-specific RNA (blue lines) fails to sufficiently concentrate TDP-43 to generate the multivalent network necessary for condensation. Scale bars, 10 μm.

Expanded View Figure Legends
Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation (2021)
Figure 1

Fig. 1. Characterization of ACVR1 responsiveness to TGF-β family ligands.

A. Parental HEK293T, HET, HOM1 and KO1 cells were treated with the indicated ligands for 1 h.

B. Cells were stimulated with increasing (doubling) doses of Activin A (0.625-20 ng/mL) for 1 h.

C. Cells were treated with BMP2 for 1 h.

D. Cells were stimulated with increasing (doubling) doses of BMP4/7 (0.625-20 ng/mL) for 1 h.

Data information: In all cases, whole cell extracts were Western blotted using the indicated antibodies. Representative experiments are shown. The levels of pSMAD1/5 relative to Actin were determined and are expressed as a fold change relative to the parental HEK293T untreated sample in A and C, or normalized to the 20 ng/mL ligand dose in parental HEK293T cells in B and D. Act, Activin A; Par, parental HEK293T cells, KO1, ACVR1 KO clone 1.