EMBO/soda-nel huggingface dataset:
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 6

Figure 6 - Wnt/Ca2+ pathway modulates vesicle transfer in primary neurons.

Top: Schematic of the culture systems used to assess cell-to-cell contact-dependent or secretion-mediated transfer of vesicles in primary neurons. After dissection, neurons were labeled in suspension with DiI (donor, D) or CTG (acceptor, A). In the co-culture system, neurons were plated in 1:1 ratio and cultured for 20 h and then treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h. In the secretion-based system, donor neurons cultured alone for 20 h were treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h, and the medium was then collected and replaced for the medium of acceptor neurons cultured separately. Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h. Bottom: Representative confocal images of co-cultured donor and acceptor neurons. The insets (right panels) show a magnification of the area depicted in the expanded field images (left). Yellow arrows point to DiI-labeled vesicles inside acceptor neurons.

Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments.

Percentage of acceptor neurons containing DiI-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons.

Confocal image showing a donor (D) and an acceptor (A) neuron connected by TNT-like structures (yellow arrowhead in the merged panel). Neurons were stained with WGA (white) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field (left).

Data information: In (A, D) scale bars represent 10 μm. (B, C) graphs represent the average of 3 independent experiments showing mean ± SEM. Statistical significance was calculated with respect to control (Ctrl); ** p ≤ 0.01, NS = not significant (one-way ANOVA).
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 7

Figure 7 - Neurons form functional TNT-like structures.

Representative confocal images of co-cultured donor (D, labeled with DiI) and acceptor (A, labeled with CTG) neurons treated or not with 200 ng/mL of Wnt5a, 200 ng/mL sFRP-2 or 200 ng/mL Wnt5a plus 200 ng/mL sFRP-2 for 4 h. Yellow arrows point to DiI-labeled vesicles inside acceptor neurons.

Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments.

Primary cortical neurons at 1 DIV were fixed and labeled with MAP-2 (green), β-III-tubulin (red) and WGA (white). The inset shows a magnified image of the area depicted in the merged panel. Yellow arrowheads point out TNT-like structures.

Transfer of α-syn fibrils is shown in neurons at 1 DIV. Cells were loaded in suspension with either Alexa-568 α-syn fibrils (red) or Alexa-488 α-syn fibrils (green) and cultured together. The insets (right panels) show a magnification of the area depicted in the expanded field image (left). Yellow arrows point to red and green α-syn puncta contained in the soma of a neuron. Yellow arrowhead points to a TNT-like connection.

A TNT-like connection containing Alexa-488 α-syn positive puncta is found in neurons at 1 DIV stained with phalloidin (white) and DAPI (blue). The insets (right panels) show the 3D reconstruction of the area depicted in the expanded field image (left).

Data information: In (A, C, D, E) scale bars in the expanded fields represent 10 µm and 5 µm in the insets. (B) graphs represent the average of 3 independent experiments showing mean ± SEM. Statistical significance was calculated with respect to control (Ctrl); * p ≤ 0.05; ** p ≤ 0.01, NS = not significant (one-way ANOVA).
Wnt/Ca2+ pathway is involved in interneuronal communication mediated by tunneling nanotubes (2019)
Figure 8

Figure 8 - Transfer of α-syn fibrils between neurons occurs through TNT-like protrusions and it can be regulated by the Wnt/Ca2+ pathway.

Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. After 20 h post-plating, neurons were treated with 200 ng/mL Wnt5a for 4 h. The insets (right panels) show a magnification of the area depicted in the expanded field (left). Yellow arrows point to α-syn puncta inside acceptor neurons.

Percentage of acceptor neurons containing α-syn puncta for each of the indicated treatments.

Time lapses of neurons loaded with Alexa-568 α-syn fibrils and cultured in control conditions or treated with 200 ng/mL Wnt5a. Neuron-to-neuron transfer of α-syn puncta (yellow arrows) through a TNT-like structure (yellow arrowheads) is shown upon Wnt5a treatment.

Percentage of acceptor neurons containing α-syn puncta in WT or βCaMKII K.O. acceptor neurons.

Representative confocal images of 1 DIV co-cultured donor (D, loaded with Alexa-568 α-syn fibrils) and acceptor (A, labeled with CTG) neurons. Neurons were obtained from WT or βCaMKII K.O. mice. Yellow arrows point to α-syn puncta inside acceptor neurons in both conditions.

Data information: In (A, C, E) scale bars represent 10 µm. In (B, D) graphs show mean ± SEM of 123-250 acceptor neurons analyzed per condition. Statistical significance was calculated with respect to control (Ctrl, in B) or WT (in D); ** p ≤ 0.01, NS = not significant (Student's t-test).
Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation (2021)
Figure 1

Figure 1. Identification of MOS Variants in Female Patients with Early Embryonic Arrest and Fragmentation.

A. Three pedigrees and four MOS variants were identified, including homozygous missense variant Asn95Lys in patient 1 (family 1), compound heterozygous missense variants Met139Thr and Arg246His in patient 2 (family 2) and homozygous nonsense variant Cys320Ter in patient 3 (family 3). The four MOS variants were inherited and identified via Sanger sequencing. WT indicates wild-type.

B. Distribution of MOS variants in genome and the corresponding amino acid sequences. Multiple sequence alignment of four segments of MOS and the orthologs, with mutated residue marked in yellow.

C. MOS variants encoding amino acid disrupted the ion pairs formed by wild-type MOS protein.

D. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar=20 µm.
Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation (2021)
Figure 2

Figure 2. The Effects of MOS Variants on Protein Expression and ERK1/2 activation in Cells and Oocytes.

A. The per million mapped reads (RPKM) values (extracted from GSE36552) showing the MOS expression level in human oocytes and early embryos (n= over 3 biological replicates).

B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.

C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells.

D. Statistical analysis of the protein expression level of FLAG, pERK1/2 and pMEK1/2 (n=2 technical replicates). The protein intensities were quantified by Image J software.

E. Diagram showing the experimental design, including procedure of MOS mRNAs injection and oocyte culture. GV oocytes were overexpressed via microinjection with wild-type MOS or MOS variant mRNAs combined with mCherry mRNAs, followed by culture for 4 h in medium containing 2.5 µM milrinone.

F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.

Data information: In A and D, data are presented as mean±SD. **P<0.01, ***P<0.001; ns., no significance (Two-way ANOVA with Tukey's multiple comparisons test in D). Detailed P value as indicated.
Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation (2021)
Figure 3

Figure 3. Oocyte MOS-ERK1/2 Pathway Inactivation Perturbs Cortical F-actin Assembly and Causes Early Embryonic Arrest and Fragmentation.

A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.

B. The relative intensities of F-actin were measured and compared among the indicated groups. The oocyte numbers for analyzed were labeled in each group. The box plot showing the F-actin intensities distribution in each group quantified using Image J (n=10-19/group).

C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm.

D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).

E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm.

F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).

Data information: For B and D, the line in the box plot represents the median value of F-actin intensity. Main box represents the values from the lower to upper quartile (25th to 75th percentile). The lower and upper whiskers indicate the min to max values. For (F) and (G), data are represented as mean±SD. One-way ANOVA with Tukey's multiple comparisons test (B) and unpaired two-tailed Student's t-test (D, F and G) were used for statistical analysis. **P<0.01, ***P<0.001; ns., no significance.
Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation (2021)
Figure 4

Figure 4. MOS Deficiency Retards Maternal mRNA Decay during Human Oocyte Maturation.

A. The Pearson correlation analysis of RNA-seq results of oocyte samples from unidentified control patients and MOSAsn95Lys patient 1.

B, C. Scatter plots showing transcriptional changes between human GV and MII stage oocytes from unidentified control patients (B) and between control and MOSAsn95Lys MII oocytes (C), separately. Genes that were increased or decreased more than two-folds are indicated in red and blue, respectively. RPKM, reads per kilobase per million mapped reads.

D. Upregulated gene numbers based on different expression level in control and MOSAsn95Lys MII oocytes.

E. Venn diagram showing the MOS-regulated decay genes, overlapped between the maturation-directed decay genes (downregulated transcripts from GV to MII oocytes from control individuals) and the upregulated genes in MOSAsn95Lys oocytes.

F. Heatmap showing the expression dynamics of MOS-regulated decay genes in GV and MII oocytes from control or MOSAsn95Lys patient.

G. The enriched biological process of upregulated MOS-regulated decay genes in MOSAsn95Lys oocytes, obtained via Gene Ontology (GO) analysis. The transcripts numbers of each biological process were labeled on the right.

H. The bar graphs show the expression levels of selected MOS-regulated decay genes (n=2 or 3 biological replicates).

Data information: In H, the RPKM values from RNA-seq results are represented as mean±SD. Unpaired two-tailed Students' t-test was used for analysis. *P<0.05, **P<0.01, ***P<0.001. Detailed P value as indicated.
Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation (2021)
Figure 5

Figure 5. ERK1/2 Inactivation Disrupts Maternal mRNA Clearance during Human Oocyte Maturation.

A. Schematic presentation of human oocyte sample collection for RNA-seq. Human immature oocytes (GVBD stage) from unidentified control women were in vitro matured with DMSO or U0126 treatment for 24 h, followed by collection of MII oocytes for RNA-seq. Ctrl means Control.

B. The Pearson correlation analysis of RNA-seq results of human oocyte samples from control and U0126 group (n=2 sample for each group). Scale bar indicates the range of the correlation coefficients (r) displayed.

C. Volcano plot of RNA-seq data obtained from human oocytes in control and U0126 group. 'Up' and 'Down' are the upregulated and downregulated transcripts in oocytes treated with U0126. The genes with upregulation and downregulation (abs[log2FC] ≥ 1; FDR ≤ 0.05) were labeled in red and blue, respectively.

D. Gene Ontology (GO) analysis of upregulated genes from RNA-seq data of U0126 group and MOSAsn95Lys MII oocytes compared with each control oocytes.

E. Venn diagram showing the upregulated genes in oocytes after U0126 treatment versus MOS-regulated decay genes (green) or upregulated genes in MOSAsn95Lys oocytes (purple). FC, fold change.

F, G. The overlapped genes between the maturation-directed decay genes and the upregulated (F) or downregulated (G) genes in U0126-treated MII oocytes. FC, fold change.

H. Heatmap showing the expression level of selected maternal genes, mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1/2 inactivation.
Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation (2021)
Figure 6

Figure 6. ERK1/2 Inactivation Causes Oocyte Mitochondrial Dysfunction during Meiotic Maturation.

A. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12).

B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17).

C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm.

D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled.

E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.

F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.

G. The graph showing the ratio of intensities of JC-1 aggregates to those of monomers in (E).

H, I. Box plot showing the ATP contents of mouse GV oocytes (H) or MII oocytes (I) from wild-type (WT) and Erk1/2oo-/- oocytes (N number was labeled in each graph).

Data information: For A, B, D and G, data represents mean±SD. For H and I, the central line in the box indicates the median value of ATP level. The lower and upper limits of main boxes represent the first and third quartiles, respectively. The lower and upper whiskers represent the limits of extreme measurements. ns., no significance, **P<0.01, ***P<0.0001 (unpaired two-tailed Student's t test).

Expanded View Figures Legends
Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination (2022)
Figure 3

Fig. 3. Ubiquitination of either of the two ID2 subunits enhances ubiquitination of the other.

A. Comparison of cryo-EM density distribution among IUbD2-DNA (Phenix-auto-sharpened map), IUbD2Ub-DNA (EMD: 21138) and ID2Ub-DNA (EMD: 21138) maps. IUbD2-DNA and ID2Ub-DNA maps, as well as IUbD2Ub-DNA model (PDB: 6VAE) were aligned to IUbD2Ub-DNA in ChimeraX. A different colour was applied for each of the protein chains of IUbD2Ub-DNA model (FANCI: slate blue, Ubiquitin-on-FANCI: green, FANCD2: cyan, Ubiquitin-on-FANCD2: magenta), while DNA was coloured red. Then each map was coloured according to nearby (within 6Å) residue colours. Contour levels were adjusted (IUbD2-DNA: 6.21, IUbD2Ub-DNA: 0.0194 and ID2Ub-DNA: 0.0162) to achieve comparable volumes among all displayed maps (ranging from 8.6 to 9.4 x 104 Å3). Arrows indicate regions of poorer density (in IUbD2-DNA and ID2Ub-DNA maps) relative to other regions of the map, as well as to equivalent positions in the other two maps.

B. Both K561 of FANCD2 and K523 of FANCI become more accessible upon ubiquitination of the other ID2 subunit. Structural comparison of relative accessibility of FANCD2-K561, upon FANCI ubiquitination (left panel), and of FANCI-K523, upon FANCD2 ubiquitination (right panel). The relative positions of these lysines upon conjugation with ubiquitin, are also shown for comparison. Residues of the other ID2 subunit within 8 Å distance from the epsilon-amino-group of the corresponding lysine are indicated as sticks. The distance to the nearest residue is shown prior and upon ubiquitination of the other ID2 subunit. In either case this increases, upon ubiquitination of the other subunit, further than 10 Å.

C-D. ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination (B), whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination (C). Protein complexes were assembled in vitro on ice in the presence of dsDNA (32 bp) and their in vitro ubiquitination at 30o C was subsequently monitored in a time-course: at indicative time-points, aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies (Top). For each protein complex, data-points from three replicate experiments (three technical replicates) were used in fitting to a one-phase association model (Bottom).
Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination (2022)
Figure 4

Fig. 4. DNA and FANCD2 protect against FANCI deubiquitination.

A. USP1-UAF1-mediated deubiquitination of V5-FANCI and FANCD2 was assessed in the absence or presence of DNA (51 bp), when ubiquitinated versions of these proteins were in isolation (IUb and D2Ub) or within singly/doubly ubiquitinated ID2 complexes (IUbD2, IUbD2Ub and ID2Ub). At indicated time-points, aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies.

B. Residual FANCI and FANCD2 ubiquitination following USP1-UAF1 treatment for 30 min at room temperature. Experiments shown in A were performed in triplicate (technical replicates), apart from ID2Ub and D2Ub deubiquitination in the absence of DNA, which were performed twice (and were thus excluded from statistical analysis). Replicate residual ubiquitination values and statistically significant changes (one-way ANOVA test with Bonferroni correction) are shown. ***p<0.001.

C. Deletion of N-terminus (∆N) of USP1 (residues 1-54) results in greatly reduced FANCD2 deubiquitination. Assays were performed in triplicate (technical replicates) as in A, but all reactions contained DNA. Left: Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies. Right: Residual FANCI and FANCD2 ubiquitination following USP1-UAF1 treatment for 30 min. Replicate residual ubiquitination values and statistically significant changes (one-way ANOVA test with Bonferroni correction) are shown. ***p<0.001.
The ubiquitin ligase parkin mediates resistance to intracellular pathogens (2013)
f1

a, Wild-type (WT) and Park2−/− BMDMs were infected with mCherry-expressing M. tuberculosis (Mtb) for 4 h and immunostained using anti-parkin antibodies. b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin. c, Quantification of ubiquitin-positive M. tuberculosis from b. Results are means ± s.e.m. of three independent experiments (**P  0.001, paired Student’s t-test). d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin. e, Quantification of ubiquitin positive M. tuberculosis from d, results are means ± s.e.m. of three independent experiments (**P  0.005, Student’s t-test). f, The parkin and actin expression in cells from e was determined by western blotting. g, Park2−/− BMDMs were transduced with lentivirus expressing BFP (−), wild-type parkin, or two separate mutant parkin alleles (T240R, P437L). Cells were infected with M. tuberculosis for 4 h and ubiquitin and M. tuberculosis co-localization was quantified and expressed relative to control BMDMs. Results are means ± s.e.m. of three independent experiments (**P  0.005, paired Student’s t-test). h, The parkin and actin expression in cells from g was determined by western blotting.
The ubiquitin ligase parkin mediates resistance to intracellular pathogens (2013)
f2

a, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained using linkage-specific ubiquitin antibodies. b, Quantification of K63 and K48 ubiquitin-positive M. tuberculosis from a. (**P  0.001, paired Student’s t-test). c, Same as a except immunostained for NDP52, p62, phospho-TBK1 or NBR1. d, Quantification of co-localization from c (n = 3 per group, **P  0.001). All errors, s.e.m.
The ubiquitin ligase parkin mediates resistance to intracellular pathogens (2013)
f3

a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a. d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001). f, BMDMs were infected with M. tuberculosis and colony-forming units (c.f.u.) at t = 0 and t = 16 h were determined by plating. Results were normalized to t = 0 (n = 3 per group, *P  0.02). g, RAW264.7 macrophages transduced with lentivirus expressing green fluorescent protein (GFP) or parkin were infected with M. tuberculosis and c.f.u. were determined after 16 h. Results were normalized to GFP-expressing cells (n = 3 per group, **P  0.0076). h, U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M. tuberculosis for 36 h and c.f.u were determined. Results were normalized to t = 0 (N = 3 per group, *P  0.02). All errors, s.e.m.