EMBO/soda-nel huggingface dataset:
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf5

(a) Knockdown of p62 expression attenuates the LC3–Dvl2 interaction. shRNA, short hairpin RNA. (b) Co-localization of GFP–LC3, p62 and Dvl in HeLa GFP–LC3 stable cells on starvation. Proteins visualized by immunofluorescence with anti-GFP (green), anti-p62 (blue) and anti-Dvl (red) antibodies. Scale bar, 10 μm. (c) p62 knockdown slows the degradation of Dvl2 on starvation. HEK-293T cells were transfected with p62 siRNA. At 48 h after transfection, cells were subjected to starvation for various times. The cell lysates were subjected to immunoblotting with the antibodies indicated. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf6

(a) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL. (b) In vitro ubiquitylation of Dvl2 by the pVHL E3 ligase complex including pVHL, Elongin B, Elongin C, Rbx1 and Cul2. In vitro-translated Flag–Dvl2 was incubated with or without purified E1, E2 and E3 ligase complex, Elongin C and pVHL. The reaction was subjected to anti-Flag immunoprecipitation. Dvl2 ubiquitylation was examined by using anti-ubiquitin immunoblotting. (c) Ubiquitylation of Dvl2 is decreased by pVHL knockdown. (d) Starvation elevates Dvl2–Cul2 interaction. After co-transfection with Myc–Cul2 and Flag–Dvl2 for 36 h, HEK-293T cells were cultured in nutrient-rich medium or starved with BFA1 (0.1 μM) for 4 h. Cells were harvested for anti-Myc immunoprecipitation followed by anti-Flag immunoblotting. Total protein expression was confirmed by immunoblotting with whole-cell lysates. (e) pVHL knockdown or UbKOGA blocks Dvl2 binding with p62 under starvation conditions. (f) Dvl2 ubiquitylation is required for its direct interaction with p62. Dvl2 was ubiquitylated by the pVHL E3 ligase complex in vitro, followed by in vitro binding assay with recombinant p62. (g) pVHL knockdown decreases Dvl2 degradation under starvation conditions. (h) Knockdown of p62 or LC3 attenuates the pVHL-promoted Dvl2 degradation under starvation conditions. Uncropped images of blots are shown in Supplementary Information, Fig. S8.
Autophagy negatively regulates Wnt signalling by promoting Dishevelled degradation (2010)
figf7

(a, b) Depletion of Beclin 1 by shRNA promotes growth in soft agar. SW480 cells stably expressing Beclin 1 shRNA or control shRNA were seeded in soft agar and treated with or without Dkk (a); the colonies were then scored after 10 days (b). Numbers in bars indicate the numbers of colonies. Results are means ± s.d. (n = 4). (Asterisk, P 0.05). Scale bar, 200 μm. (c) Representative images from colon cancer tissues by immunohistochemistry staining. Negative correlation between Beclin1 and Dvl was observed in late stages of tumours, whereas positive correlation between p62 and Dvl in late stages of tumours. Scale bar, 25 μm. (d) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies. N, normal tissue; T, tumour. (e) Working model of autophagic degradation of Dvl2.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 1

miR-155 expression is induced after mycobacterial infection.

(A) miR-155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB/c mice 6 weeks postinfection. (B) Murine bone marrow-derived macrophages (BMDMs) were infected with BCG at an MOI of 5 for the indicated times, and the expression levels of miR-155 were measured by real-time PCR. (C and D) RAW264.7 cells were infected with BCG at an MOI of 5 for the indicated time points (C) or at indicated MOIs for 24 h (D). The expression levels of miR-155 were examined by real-time PCR. (E and F) RAW264.7 cells were infected with H37Ra at an MOI of 5 for the indicated time (E) or at indicated MOI for 24 h (F). The expression levels of miR-155 were examined by real-time PCR. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 2

miR-155 decreases mycobacterial survival in macrophages.

(A–C) RAW264.7 cells were transfected with control or miR-155 mimic for 24 h followed by BCG (A), H37Ra (B) or H37Rv (C) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. (D–F) RAW264.7 cells were transfected with control or miR-155 inhibitor for 24 h followed by BCG (D), H37Ra (E) or H37Rv (F) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. (G and H) RAW264.7 cells were transfected with miR-155 mimic (G) or inhibitor (H) for 24 h followed by Texas Red-labeled BCG infection at an MOI of 10 for 1 h. Phagocytosis of mycobacteria was determined by flow cytometry. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 3

miR-155 promotes mycobacterial phagosome maturation.

RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A), or labeled with a fluorogenic substrate for proteases, DQ-Green (C). The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B) or DQ-Green labeled (D) lysosomes was quantified, respectively. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with lysosomes; scale bar = 5 µm. Quantification of data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 4

miR-155 induces autophagy in macrophages.

(A–D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B). The LC3 levels were detected by Western-blot, and the ratios of LC3-II/β-actin were calculated as shown below the representative blot (C and D). (E and F) RAW264.7 cells transfected with miR-155 mimic (E) or inhibitor (F) was incubated with DMSO or bafilomycin A1 (BafA.) at a concentration of 100 nM for 2 h, and then LC3 levels were detected by Western-blot. The ratios of LC3-II/β-actin were calculated as shown below the representative blot. ***, p<0.001.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 5

miR-155 promotes autophagosome formation.

(A and B) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic for 24 h, and GFP-LC3 puncta (>1 µm) were detected by confocal microscopy (A) and quantified (B). RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green), and LC3 puncta (>1 µm) were detected by confocal microscopy (C) and quantified (D). (E and F) RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with a specific fluorescent dye MDC (50 µM). The MDC-labeled autophagic vacuoles were detected by confocal microscopy (E) and cells with MDC-positive autophagic vacuoles were quantified (F). Cells treated with rapamycin were used as a positive control. Arrows indicate the GFP-LC3 puncta or autophagic vacuoles labeled with MDC; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 300 cells). **, p<0.01; ***, p<0.001.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 6

miR-155-induced autophagy promotes the formation of mycobacterial autophagosomes.

(A) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG with LC3 was detected by confocal microscopy. (B) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown. (C) RAW264.7 cells were transiently transfected with control or miR-155 mimic, and then infected with Texas Red-labeled BCG for 1 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green). The co-localization of BCG with endogenous LC3 was detected by confocal microscopy. (D) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown. (E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 7

miR-155-induced autophagy promotes the elimination of intracellular mycobacteria.

(A and B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with DMSO or 3-MA for 2 h. Protein levels of LC3 were detected by Western-blot in uninfected cells (A). Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h (B). (C and D) RAW 264.7 cells were transiently co-transfected with control or miR-155 mimic together with a control siRNA or Atg7 siRNA. The expression levels of Atg7 and LC3 were detected by Western-blot (C). Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h (D). Values of LC3-II/β-actin ratios are indicated below the representative blot. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 8

miR-155 post-transcriptionally represses Rheb expression by targeting its 3′UTR.

(A) Sequences of mouse and human miR-155 and their predicted interactions with conserved 7-mer 1A miR-155 seeds found within the Rheb 3′UTRs of different species are shown. The sequence of the Rheb 3′UTR seed mutant used for the reporter assays and the predicted disruption of the miR-155 interaction are also shown. (B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant. (C–F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D), and expression levels of Rheb mRNA were detected by RT-PCR (E and F).
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb. (2013)
Figure 9

miR-155-induced autophagy promotes the co-localization of BCG with autophagosomes by suppressing Rheb expression.

(A) RAW264.7 cells were transfected with a plasmid expressing Rheb for 24 h, and then incubated with DMSO or BafA. for 2 h. The expression levels of Rheb and LC3 were detected by Western-blot. Values of LC3-II/β-actin ratios are indicated below the representative blot. (B and C) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h, and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG and MDC-labeled autophagosome was detected by confocal microscopy. Arrows indicate Quantification of BCG co-localization with autophagosomes described in B is shown (C). (D) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h. Cells were infected with BCG for 1 h, and washed for three times to remove extracellular mycobacteria. Intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.
Molecular crowding and RNA synergize to promote phase-separation, microtubule interaction, and seeding of Tau condensates (2022)
Figure 3

Figure 3. Phosphorylation modulates Tau condensation and condensate affinity to surfaces.

Recombinant phosphorylated Tau expressed in insect cells (p-Tau) de-mixes in the presence of PEG but fails to coacervate with RNA (5 μM p-Tau, 10% PEG). In the presence of PEG, polyA-Cy3 co-condensates with p-Tau (arrow heads). Scale bar=10 μm.

Tau phospho-variants generated by in vitro phosphorylation of non-phosphorylated Tau from E. coli with different kinases (PKA, GSK3β, or Cdk5) show differences in phosphorylation at specific phospho-site (pT205, pS262/pS356, pS324 and pS404) tested by Western blot. Non-phosphorylated Tau and p-Tau from insect cells are loaded for comparison. Position of tested p-sites is indicated in the Tau sequence cartoon. Western blot for total Tau (Tau13 antibody) confirms equal loading (0.2 μg Tau per lane) of the Tau preparations.

Phos-tag gel shows degree and heterogeneity of phosphorylation in the different Tau phospho-variants. Intensity along the lanes are plotted as colored lines next to gel image.

Condensation of Tau phospho-variants in the Tau:PEG, Tau:RNA (polyA), and pTau:PEG:RNA systems was determined by turbidity measurements for condensates in solution (absorption at 600 nm) and by microscopy for surface attached condensates (surface coverage; top left). Turbidity measurements are shown on the left y-axis (blue circles), and surface coverage in % on right y-axis (black data points). Scale bars=20 μm. N=4 experiments, data points represent the average of 5 data points from turbidity measurements taken at 20±5 min after LLPS induction. Top right panel shows turbidity measurements of Tau alone as negative control for LLPS detection. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Molecular crowding and RNA synergize to promote phase-separation, microtubule interaction, and seeding of Tau condensates (2022)
Figure 5

Figure 5. Tau coacervates only percolate in the presence of PEG.

FRAP experiments of Tau:PEG, Tau:polyA, and Tau:PEG:polyA condensates at 1h, 4h, and 24h after LLPS induction. Tau:PEG and Tau:PEG:polyA condensates show a decrease in recovery rate and mobile fraction over time. Tau:polyA coacervates maintain fast recovery and a large mobile fraction even after 24h. Time until half maximum recovery: t(y1/2)polyA: 1h≈2s, 4h≈3.5s, 24h≈4.5s; t(y1/2)PEG: 1h≈6s, 4h≈8s, 24h≈11s; t(y1/2)PEG+polyA: 1h≈4.5s, 4h≈9.5s, 24h≈9s). Scale bars=5 μm for Tau:polyA and Tau:PEG, and 10 μm for Tau:PEG:polyA. Data presented as error bars (SEM), N=15 to 20 condensates per condition and time point.

Microscopy of Tau:PEG:polyA condensates (Tau-Dylight488 and polyA-Cy3) shows condensates with different Cy3 intensities. The bimodal fluorescence intensity distribution suggests two groups of condensates with high or low Cy3 fluorescence. Cy3-intensities, normalized to Tau-DyLight488 intensities in the same condensates, were independent of condensate size. Scale bar=10 μm.

FRAP of Tau-Dylight488 and polyA-Cy3 of 4h-old Tau:PEG:polyA condensates. The mean FRAP of polyA-Cy3 is lower than of Tau-Dy488 (left). Data shown as mean±SEM, n=24 condensates. FRAP data of individual condensates (bottom; #1 to #3) shows a decrease of Tau=Dy488 (middle) and polyA-Cy3 (right) FRAP with an increase of polyA-Cy3 (relative to Tau-Dy488) in the condensates. Scale bar=5μm.
Molecular crowding and RNA synergize to promote phase-separation, microtubule interaction, and seeding of Tau condensates (2022)
Figure 6

Figure 6. Tau coacervates and condensates produce Tau species with seeding potential.

SDS-PAGE of Tau incubated in LLPS (PEG, tRNALLPS, PEG:tRNALLPS) or aggregation (hepAGG) conditions for 1h, 24h, or 72h. SDS-stable Tau dimers and oligomers species evolve in LLPS and hepAGG samples after 24h. After 72h, all conditions contain SDS-stable Tau oligomers and dimers.

Thioflavin-T assay for the enhancement of in vitro Tau aggregation by aged Tau condensates. TauΔK280 (10 µM) incubated with heparin (molar ratio Tau:heparin=4:1; [TauΔK280]AGG) was used as base line condition for Tau aggregation. Addition of 24h-old or 72h-old Tau LLPS samples (9.2 µg/ml=0.2µM Tau) produced an increase in TauΔK280 aggregation for all LLPS conditions (PEG, tRNA, PEG:tRNA) and hepAGG. Addition of Tau only did not affect TauΔK280 aggregation. Data shown as mean±SD, n=3. One-way ANOVA with Tukey test for multiple comparison for values at 30 min. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

HEK sensor cell Tau aggregation assay. Cells were treated with 24 h-old condensates or aggregates (1.2 µM Tau in the presence of 0.8% lipofectamine). The number of Tau aggregates was determined from low magnification (20x) images taken in the green channel and normalized to the number of cells (DAPI). Scale bar=100 μm.

Quantification of seeding assay in (C). Data were collected from three images of 3 wells for each condition. Data shown as mean±SD, N=3 experiments, 1-3 images per experiment and condition. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Cell toxicity (LDH assay) induced by treatment of HEK sensor cells with condensates or aggregates. N=3 experiments. Data shown as mean±SD. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Molecular crowding and RNA synergize to promote phase-separation, microtubule interaction, and seeding of Tau condensates (2022)
Figure 8

Figure 8. FRAP and FLIM reveal differences in molecular packing in Tau accumulations.

FRAP of Tau accumulations show no recovery of NUC Tau (NUC: <10% recovery; t1/2 ≈11 s), ~20% recovery of CYT and NE Tau with a faster recovery rate for NE Tau (NE: t1/2 ≈2.5 s; CYT: t1/2 ≈7 s). N=15-20 Tau accumulations per type. Data shown as mean±SD.

Example FRAP image series for cytosol and the different Tau accumulation types in HEK sensor cells treated with AD brain lysate (pre-bleach=-2s, immediately after bleaching=0s, after recovery=40s). Arrows indicate position of bleached ROIs. Scale bars=5 or 10 μm as indicated.

High-resolution CFP FLIM of HEK sensor cells expressing CFP/YFP-TauRDP301L and treated with Tau condensates. FRET between CFP and YFP induces CFP-lifetime reduction in densely packed CFP/YFP-TauRDP301L accumulations. Fit-free analysis of the lifetime distribution in phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, LT-4=fully aggregated CYT Tau. LT-1 corresponds to soluble CFP-TauRD in the cytosol of cells without accumulations. Asterisks indicate position of individual cell nuclei. Scale bars=10 μm.

CFP-FLIM of seeded HEK sensor cells expressing only CFP-TauRDP301L. The dense packing of CFP-TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP-lifetime. Fit-free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, and LT-1=soluble CFP-TauRD in the cytosol of cells without accumulations. Graphs show lifetimes of CFP-lifetime components assigned to soluble, NE, and CYT+NUC Tau in 6 different images (left graph, RM one-way ANOVA with Tukey post-test) and lifetime reduction relative to LT-1 in each respective image (right graph, one-way ANOVA with Tukey post-test.). N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. Scale bars=10 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FLIM of 1h- and 24h-old CFP-Tau (full-length) condensates and heparin induced CFP-Tau aggregates. Lifetime components are indicated in phasor plots. Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP-Tau. N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. One-way ANOVA with Tukey post-test. Scale bar=5 μm.­ *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Bottom right: Schematic of lifetime quenching in condensates and aggregates.