EMBO/soda-nel huggingface dataset:
Mitochondrial-nuclear co-evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins (2016)
Figure 2

Figure 2. CCM1 incompatibility results in reduced levels of 15S rRNA and mtDNA-encoded proteins.

(A) Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto) or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which were probably due to some unknown protein modifications. (B) The level of 15S rRNA is reduced in Sc+Sb-CCM1 cells. RNA isolated from Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots. (C) Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. (D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. (E) The steady-state level of mtDNA-encoded proteins is reduced in Sc+Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2 and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein.
Mitochondrial-nuclear co-evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins (2016)
Figure 3

Figure 3. Incompatibility of Sb-CCM1 can be relieved by mutations in the mitochondrial genome.

(A) Tetrad analysis reveals that the respiration defect of Sc+Sb-CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes. The suppressor clones were crossed with the Sc+Sb-CCM1-ρ⁰ strain and the tetrads from the cross were analyzed. A non-Mendelian 4:0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA (left panel). Otherwise, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation (right panel). (B) Partial sequence alignment of 15S rRNA from S. bayanus, S. cerevisiae and two mitochondrial suppressor clones (#1 and #2) of Sc+Sb-CCM1. A single-nucleotide deletion at position 188 and four nucleotide substitutions at positions 1524-1527 found in both suppressor clones are labeled in red.
Mitochondrial-nuclear co-evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins (2016)
Figure 4

Figure 4. Changes in the PPR domain of Sb-CCM1 rescue the incompatibility.

(A) Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc+Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae. (B) Sequence alignment shows that most of the Sb-CCM1 suppressor mutants contain de novo amino acid changes and only two mutants have switched their residues from the S. bayanus sequence to the S. cerevisiae sequence (F294L and D400N). Letters beside the phylogenetic tree (in which the line lengths are not proportional to the evolutionary distances) indicate the amino acid state at the 400th residue and parentheses contain the codons encoding for the amino acids. (C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus. (D) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs. The predicted protein structure of Ccm1 is shown at left. Blue and green colors indicate the two α-helices in each predicted PPR motif and white color indicates additional α-helices in the N-terminus and the middle region. Red color indicates the positions of the suppressor mutations. Predicted PPR motifs occupy around 52% of the whole protein (453 of 871 residues). The box at right displays a magnified version of some PPR motifs where the substitutions are located.
Mitochondrial-nuclear co-evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins (2016)
Figure 5

Figure 5. Suppressor mutations partially restored the levels of 15S rRNA and mtDNA-encoded proteins.

(A) 15S rRNA levels are increased in the suppressor clones. Total RNA was isolated from the replacement strains (Sc+Sc-CCM1 and Sc+Sb-CCM1), two nuclear suppressor strains (Sc+Sb-CCM1D400N and Sc+Sb-CCM1E375Q) and one mitochondrial suppressor strain and then subjected to quantitative PCR analyses. Relative mRNA abundance was calculated by normalizing the 15S rRNA level to the ATP6 mRNA level. Graph was plotted using data from five independent repeats for each strain and p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. **, p value < 0.01. Asterisks in black, comparing Sc+Sc-CCM1 with others. Asterisks in gray, comparing Sc+Sb-CCM1 with others. (B) The steady-state level of mtDNA-encoded proteins is increased in the suppressor clones. Protein extracts from the replacement and suppressor strains were hybridized with antibodies against G6PDH, Cox1, Cox2 or Cox3.
A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis (2022)
Figure 2

Figure 2: Basal NSCs in the adult SVZ do not express Nestin and do not extend primary cilia.

(A) Representative confocal photomicrographs showing coronal slices from 8W hGFAP‐tTA;H2B‐GFP mice with and without doxycycline treatment, upon immunofluorescent staining with the stem cell marker Nestin (red). DAPI was used for nuclear counterstain. Cross indicates slice directions: M = medial, D = dorsal, V = ventral, L = lateral. Scale bar = 20 µm.

(B) Quantification of Nestin expression in apical and basal cells in doxycycline-treated and untreated animals. N = 3 (- Doxy), N = 4 (+ Doxy).

(C) Same as (A), with addition of the primary cilia marker AC3 (yellow). Arrows indicate Nestin-positive (white) and GFP-positive (green) cells with primary cilium. Scale bar = 10 µm.

(D) Quantification of ciliated GFP+ NSCs in apical and basal cell layer as percentage of total apical/basal GFP+ cells. N = 7.

(E) Quantification of primary cilia on cells in the apical or basal cell layer according to stem cell marker; N = 7.

Data information: N number refers to biological replicates; for each biological replicate, at least three technical replicates were performed. Data is represented as mean ± SEM. * and # indicate significance: between apical and basal (*) and between doxycycline treatments (#); */#p<0.05, **p<0.01, by one-way ANOVA with Tukey's multiple comparisons test (B) or two-tailed unpaired student's t-test (D).
A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis (2022)
Figure 3

Figure 3: Apical and basal NSCs display stemness.

(A) Repesentative confocal photomicrographs of coronal sections of the SVZ of 8W hGFAP-H2BGFP mice, which had been administered doxycycline (Doxy) as indicated. Nuclear GFP expression and Ki67 immunoreactivity are shown in green and red, respectively. DAPI counterstaining of the nuclei is blue. Scale bar indicates 20 µm.

(B, C) Quantitative analyses of the number of apical and basal cells expressing GFP (G+) or not (G-); N = 8 (B), and Ki67 immunoreactivity in these populations; N ≥ 4 (C).

(D) Quantitative analysis of the number of label retaining apical and basal G+ cells in mice injected with IdU at embryonic day E14 or E18 and analysed 8W after birth. N = 4.

(E-F) Quantification of the percentage of apical and basal G+ cells expressing intermediate (Gi) or high (Gh) GFP levels with or without doxycycline (Doxy) treatment upon FACS analysis; N ≥ 21 (E) and in brain slices; N = 7 (- Doxy), 9 (+ Doxy; F).

Data information: N number refers to biological replicates; for each biological replicate (except E), at least three technical replicates were performed. Bars represent mean ± SEM. * indicates significance between doxycycline treated and nontreated specimens with apical or basal cell populations. * p<0.05, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test.
A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis (2022)
Figure 5

Figure 5: Apical stem cells give rise to basal neural stem cells.

(A) Representative micrographs of coronal sections of WT mice intraventricularly injected with AAV-GFP, and stained for GFAP and SOX9 at 14 days after injection. Scale bar indicates 30 µm. Arrows indicate apical GFP+ (white) and basal GFP- (green) NSCs labelled by SOX9 and GFAP.

(B) Representative micrographs of the OB of AAV-GFP-injected animals, at 14 days (14D) and 6 weeks (6W) after virus injection. Scale bar indicates 20 µm.

(C) Quantification of GFP+ and/or DCX+ cells in the OB, as percentage of total DAPI cells N ≥ 5.

(D-E). Representative confocal images of coronal slices of the core of the olfactory bulb (OB) obtained from RiboTag mice injected with AVV-hGFAP-mycCRE particles. In (D) mice were analysed 8 weeks after injection in the lateral corner of the V-SVZ. Arrow in point at Doublecortin (DCX) immunopositive neuroblasts displaying hemagglutinin-tagged ribosome (RiboTag-HA). In (E) mice were analysed 16 weeks after injection in the lateral ventricle or in the lateral corner as indicated. Scale bars = 20 µm.

(F) Quantification of RiboTag-HA (HA)+ and/or DCX+ cells in the OB in N ≥ 2.

(G) Number of apical and basal Eh cells counted by flow cytometry and given as percentage of total G+ cells in Virgin (Virg) and littermates dams that had been lactating for 7 days. N ≥ 5.

Data information: N number refers to biological replicates; for each biological replicate (except G), at least three technical replicates were performed. Bars represent mean ± SEM.* indicates significance: *p<0.05, determined by two-way ANOVA with Sidak's multiple comparisons test.
A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis (2022)
Figure 7

Figure 7: Interference with the Notch signalling pathway leads to increased proliferation.

(A, B) Representative micrographs of the V-SVZ of mice injected with an AAV overexpressing GFP and a shRNA against Hes1 (AAV Hes1-sh) or a scrambled sequence (AAV sc-sh). Coronal sections were stained with either notch intracellular domain (NICD, A) or Ki67 to visualize cycling cells (B). DAPI was used for nuclear counterstain, scale bars indicate 20 µm. Arrows indicate apical GFP+ (white) and basal GFP- (yellow) cells displaying NICD staining.

(C-E) Quantification of fold change in NICD-levels; N = 3 (C), Ki67+ cells; N = 5 (D) and GFP+DCX- cells in the olfactory bulb (OB) 14 days (14D) and 6 weeks (6W) after virus injection (E).

(F-H) Quantitative analysis illustrating NICD levels in apical and basal cells in virgin (Virg) and littermate dams that have been lactating for 7 days; N = 11 (F), the total percentage of apical G+ and G- cells (G) and the percentage of activated cells in each population after 17 days of lactation. N = 5 (H).

Data information: N number refers to biological replicates; for each biological replicate (except G+H), at least three technical replicates were performed. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, determined by two-way ANOVA with Sidak's multiple comparisons test (C-F) or two-tailed unpaired student's t-test (G+H).
Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance (2022)
Figure 1

Figure 1

Sybodies Sb#15 and Sb#68 bind non-overlapping epitopes on the spike protein, and inhibit ACE2 binding. (A) Affinity determination of Sb#15 and Sb#68 against the immobilized spike protein (S-2P) using GCI. The data were fitted using a heterogeneous ligand model. (B) Left, GCI epitope-binning experiment showing Sb#15 (blue), Sb#68 (red) and their combination (black) against immobilized spike protein (S-2P). Since both sybodies were present at saturating concentrations, the increased amplitude is indicative of simultaneous binding. Right, ELISA experiment confirming dual-binding of Sb#15 and Sb#68. Myc-tagged Sb#15 was immobilized on an anti-myc antibody-coated ELISA plate, followed by exposure of biotinylated RBD, which was pre-mixed with tag-less sybodies (indicated on the x-axis). (C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.
Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance (2022)
Figure 2

Figure 2

Neutralization of viral entry using pseudotyped VSVs. Neutralization assays using VSVΔG pseudotyped with wildtype SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. (A) Sb#15, Sb#68, or an equimolar mixure of both sybodies. (B) Sb#14, Sb#16, Sb#45, or, as a positive control, the previously-described sybody MR3. (C) Neutralization by bivalent sybody-Fc fusions. Error bars correspond to standard deviations of three biological replicates.
Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance (2022)
Figure 4

Figure 4

Fusion of Sb#15 and Sb#68 into biparatopic and multivalent molecules strongly enhance binding affinity and neutralization. (A) Schematic drawing of biparatopic GS4 and multivalent Tripod-GS4r constructs. (B) Affinity determination of GS4 against immobilized spike protein using GCI. (C) Neutralization assay using VSVΔG pseudotyped with wildtype Spike-protein. Relative infectivity was determined in response to increasing GS4 or Tripod-GS4r concentration. The corresponding neutralization data for isolated Sb#15 and Sb#68 are provided as reference. (D) Control experiment using VSVΔG pseudotyped with VSV-G. Infectivity was not affected by Tripod-GS4r. Error bars correspond to standard deviations of three biological replicates.
Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance (2022)
Figure 5

Figure 5

The bispecific construct GS4 mitigates emergence of novel escape mutants. (A) Structural context of RBD escape mutants resulting from adaptation experiments in the presence of either Sb#15 or Sb#68 alone (blue spheres). Prominent globally-circulating mutations are shown as salmon spheres. (B) GCI-based kinetic analysis of the purified RBD bearing either no mutation (WT), or identified escape mutations Q493R or P384H. Left, middle, and right plots correspond to immobilized Sb#15, Sb#68, or GS4, respectively. (C) Neutralization assay using VSVΔG pseudotyped with Spike-protein containing the Q493R or P384H mutation, respectively. Relative infectivity in response to increasing binder concentrations was determined. Error bars correspond to standard deviations of three biological replicates.
The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. (1997)
Figure 7

ASGP-R labeling in FP+ and endosomes. Cells exposed for 40 min to 5-nm gold tracer. Section immunolabeled for ASGP-R with 10-nm gold. (a) Low ASGP-R labeling in tracer-carrying endocytic vesicles (arrowheads), adjacent to (top) or fusing with (bottom) an AVi/d. (b) ASGP-R labeling at the plasma membrane and in endosomal elements near the cell surface (ex, extra cellular space). Vesicles of the MVE (asterisk) and VE (arrowhead) type, containing endocytosed gold, are present in the same area. (c) As b. A VE-like vesicle with endocytosed gold and low ASGP-R labeling (arrowhead) is shown connected to an ASGP–R rich element (arrow), possibly a recycling endosome. Bars, 200 nm.
The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. (1997)
Figure 2

Labeling patterns of cathepsin D and CAIII in MU picked-up cryosections. (a) Cells treated as in Fig. 1 b, but the section was immunolabeled for cathepsin D, which is not detectable in the AVi, moderately present in the AVi/d, and abundant in AVd. Cells in b and c had endocytosed 5-nm gold tracer for 20 and 40 min, respectively, before fixation and CAIII labeling. (b) Complex fusion profile of a CAIII-positive AVi, an AVd which is without CAIII labeling, and a VE that contains an endocytosed gold probe (arrow). Close to the AVd is another tracer-bearing VE. (c) Fusion profile of a tracer-bearing MVE and an AVi/d that exhibits a single limiting membrane and a CAIII-labeling concentration similar to that of the cytoplasm. Bars, 200 nm.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 1

Ubiquitinated aggregates accumulate near SCVs in HeLa cells.

(A) Single confocal sections of HeLa cells infected with GFP-expressing S. Typhimurium strains (blue) for 10 h, fixed and immunolabelled for LAMP1 (green) and ubiquitin (Ub, red) (false coloured, scale bars, 5 µm). The far right panels show merged images of LAMP1, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitinated aggregates. (B) Quantification of the percentage of infected cells containing SCV-associated ubiquitinated aggregates. Cells were processed as in (A) and analysed by fluorescence microscopy. (C) Kinetics of the formation of SCV-associated ubiquitinated aggregates. Cells were infected with GFP-expressing S. Typhimurium strains for the indicated amount of time, and processed as in (A). (D) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates in HeLa cells infected with S. Typhimurium strains for 14 h. Cells were processed as in (A) and analysed by fluorescence microscopy. For all quantifications, a minimum of 50 infected cells were counted per experiment and values are the mean ± SEM from at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 2

Ubiquitin accumulates in dense cytosolic aggregates and forms K63-linked chains.

(A) Immunoelectron microscopy of HeLa cells infected with S. Typhimurium strains for 12 h (ubiquitin - 10 nm gold particles; scale bars, 0.5 µm). Arrows indicate ubiquitin accumulations near bacteria within vacuoles. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 10 h and immunolabelled for ubiquitin (all chain types - Ub, red) and lysine-48-linked (K48-Ub, green) or lysine-63-linked (K63-Ub, green) ubiquitin chains (false coloured, scale bars, 5 µm). Arrows indicate SCV-associated ubiquitin and K63-Ub labelling.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 3

SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to <italic>Salmonella</italic> microcolonies.

Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3. (C–E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 4

SCV-associated ubiquitinated aggregates are autophagy substrates.

(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001. (B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.
The Salmonella deubiquitinase SseL inhibits selective autophagy of cytosolic aggregates. (2012)
Figure 5

SseL reduces the accumulation of ubiquitinated aggregates and ALIS in macrophages.

(A) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (green) for 10 h and immunolabelled for ubiquitin (Ub, red) (scale bars, 5 µm). (B) Quantification of infected macrophages containing ubiquitinated aggregates 10 h after bacterial uptake. Cells were processed as in (A) and analysed by fluorescence microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of at least 3 independent experiments. * p<0.05; *** p<0.001. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. (C) Immunoelectron microscopy of RAW264.7 macrophages infected with the ΔsseL mutant bacteria for 12 h. Arrows and arrowheads indicate ubiquitin (15 nm gold particles) and p62 (10 nm gold particles), respectively (scale bar, 0.5 µm). (D and E) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (blue) and immunolabelled for ubiquitin (Ub, red) and (D) p62 (green) or (E) LC3 (green). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3 (scale bars, 5 µm). Cell outlines and nuclei are delineated by white lines.