EMBO/soda-nel huggingface dataset:
Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy (2018)
Figure 3

Figure 3 BAK is ubiquitinated in response to various mitophagy stimuli and by endogenous Parkin, but is compromised in Parkinson's Disease-associated Parkin mutants.

(A) HeLaParkin cells were treated with CCCP (10 μM), antimycin A and oligomycin (AO) for 2h or GTPP (10 μM) for 6h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/- SD.

(B) SH-SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN (sgPRKN) or a non-targeting control (sgCon) were assessed for Parkin expression by immunoblotting of cytosolic fractions.

(C) SH-SY5Y cells generated in (B) were treated with antimycin A and oligomycin (AO) for 3 h prior to cell lysis and immunoblotting. Mono and di-ubiquitinated BAK in Parkin-expressing cells following AO are indicated (asterisk). Data is representative of three independent experiments.

(D) Domain architecture of human Parkin with positions of selected PD-associated mutations. Ubl, ubiquitin-like; IBR, in-between-RING (Gladkova et al., 2018). HeLa cells expressing wild-type Parkin or the indicated PD-associated Parkin mutants were treated with antimycin A and oligomycin (AO) for 2h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins in AO-treated samples relative to untreated control from three independent experiments. Error bars represent mean +/- SD. All Parkin constructs were N-terminally FLAG-tagged except R104W.
Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy (2018)
Figure 4

Figure 4 Oligomerisation of BAK restricts its ubiquitination and induces Parkin activity.

(A) BN-PAGE and SDS-PAGE of HeLa+HA-Parkin cells treated with 1 µM of each of ABT-737 and S63485 for 3 h (BH3), antimycin A and oligomycin (AO) for 2 h or a combination of the two. Experiment was performed in the presence of 10 µM QVD.oph and immunoblots are representative of three independent experiments.

(B) UBA enrichment of ubiquitinated proteins from HeLa+HA-Parkin cells, BAX-/- BAK-/- (DKO) HeLa cells or wild-type (WT) HeLa cells expressing HA-Parkin in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of QVD.oph (10 μM). Representative of three independent experiments.

(C) HeLa cells expressing HA-Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non-targeting sgRNA control, treated with AO (2 h) to stabilise PINK1 expression and immunoblotted for PINK1.

(D) Parkin activity induced by apoptotic mitochondrial damage is PINK1-dependent. UBA enrichment of ubiquitinated proteins from HeLa cells generated in (C) in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of Q-VD.oph (10 μM). *, ubiquitinated protein. Representative of two independent experiments.

(E) Schematic showing Parkin ubiquitination of BAK monomer in non-apoptotic cells, but not the BAK homo-dimer in cells undergoing apoptosis. In response to mitophagy stimuli, PINK1 phosphorylates ubiquitin to recruit Parkin, which in turn is phosphorylated by PINK1 to become activated to ubiquitinate monomeric BAK on K113. Mitochondrial outer membrane (MOM) permeabilisation driven by BAK oligomers during apoptosis provokes Parkin activity in a PINK1-dependent manner. Although Parkin can ubiquitinate mitochondrial outer membrane substrates (e.g. Mfn2) to promote mitochondrial clearance, it cannot ubiquitinate dimerised BAK on K113.
Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy (2018)
Figure 5

Figure 5 Ubiquitin-conjugated BAK at position 113 delays BAK permeabilisation of liposomes by impairing homo-oligomerisation.

(A) Schematic of fluorescence-based liposome assay to measure BAK activation on model membranes.

(B) Non-reducing SDS-PAGE and immunoblot of recombinant BAK and BAK-Ub following treatment with 25 mM TCEP.

(C) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID-BH3 peptide to 50 nM BAK or BAK-Ub in the presence or absence of 25 mM TCEP. Data is mean +/- SD of three independent experiments.

(D) BN-PAGE of BAK and BAK-Ub on liposomes following incubation with 4 µM BID-BH3 peptide in the presence or absence of 25 mM TCEP, immunoblotted for BAK. Data representative of three independent experiments. Graphs shows densitometric analysis of the BAK tetramer (4x) from three or two independent experiments. Error bars represent mean +/- SD for n=3 or mean and range for n=2.
Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy (2018)
Figure 6

Figure 6 Ubiquitination of BAK on mitochondrial membranes and in cells inhibits its ability to mediate mitochondrial outer membrane permeabilisation and apoptosis.

(A) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK.
(B) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function. Mitochondrially-enriched membranes from MEFs expressing BAK or BAK K113C were cross-linked (CuPhe) with UbG76C and treated with cBID for 30 min at 30°C. Supernatant (S) and pellet (P) fractions were then immunoblotted for BAK under non-reducing conditions, or cytochrome c or ubiquitin under reducing conditions. Representative of three independent experiments. Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS-PAGE.

(C) Parkin-mediated ubiquitination limits BAK activation. HeLa+HA-Parkin cells were treated with antimycin A and oligomycin (AO) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT-737 and S63485 (BH3 mim) for 1 h. Intracellular flow cytometry was performed using the conformation-specific anti-BAK antibody G317-2. Representative histograms are shown and the fold increase in mean fluorescence intensity (MFI) is plotted from three independent experiments with error bars showing SD. Data are normalised to untreated control.

(D) Parkin-mediated ubiquitination limits BAK apoptotic activity. BAK-/-BAX-/- HeLa cells expressing Parkin and BAK were treated with AO (2 h) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction. Error bars represent mean +/- SD.

(E) Parkin activity limits cytochrome c release. HeLa cells or HeLa cells expressing Parkin were treated and analysed as in (C). Immunoblots are representative of three independent experiments. Ubiquitinated BAK indicated (arrow). Graph shows densitometric analysis of three independent experiments showing cytochrome c release (supernatant/supernatant+membrane) relative to 100 nM cBID as 100% release. Error bars represent mean +/- SD.
Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II (2021)
Figure 1

Figure 1.

Sister kinetochores individualize already in anaphase I.

A) Oocytes were fixed for chromosome spreads 8 hours after GVBD, and stained for PP2A-c (red), Sgo2 (green), CREST (blue) and DNA (Hoechst, shown in grey). Spreads were classified into metaphase I and anaphase I, depending on whether chromosome segregation had taken place, or not. Arrowhead indicates fused sister kinetochores, arrows indicate separated sister kinetochores.

B) Example of a whole mount oocyte staining, in early anaphase I after cold-treatment to visualize only cold-stable microtubule fibers, stained with anti-tubulin antibody (green). Kinetochores are stained with CREST serum (red) and chromosomes with Hoechst (blue). Shown is an overlay of 20 z-sections. Arrows indicate separated sister kinetochores.

C) Metaphase I (6 hours after GVBD) and metaphase II (16-20 hours after GVBD) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin (pink) and the centromere (green), respectively. Chromosomes were stained with Hoechst (blue). Below, schematic representation of a bivalent in metaphase I and a dyad in metaphase II. Enlargement shows the centromere region and a region that we call "chromatid junction" with the corresponding scheme.

D) Quantification of total Sgo2 signal at the centromere and the chromatid junction, normalized to CREST at centromeres, on chromosome spreads from oocytes at the indicated stages of meiotic maturation. Each dot shows mean per oocyte; the number of oocytes analyzed is indicated. Below: scheme illustrating how Sgo2 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines, see Material and Methods for quantification approach).

E) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated.

F) Quantification of E) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair (dot plot on the left), and distance between sister kinetochores (dot plot on the right) at the indicated stages of meiosis I. Meta: metaphase, ana: anaphase, telo: telophase. Each dot represents one kinetochore pair; the number of kinetochore pairs analysed is indicated. Below: scheme of Sgo2 signal (in red) measurements.

Data information:

On each graph mean is shown, error bars are ± SD, asterisks indicate significant difference (****=p<0,0001) according to Mann-Whitney U test. AU: arbitrary units. n indicates number of analysed kinetochores, kinetochore pairs or oocytes, as indicated. Scale bars, 10μm. See also Fig EV1.
Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II (2021)
Figure 2

Figure 2.

Sgo2 localized by Mps1 to the chromatid junction is dispensable for sister chromatid segregation.

A) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Sgo2 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). On the right, schemes illustrate the chromosome figures observed, and the corresponding Sgo2 staining (in red).

B) Dyads of metaphase II chromosome spreads of control, Bub1KD, Mps1∆N and Reversine-treated oocytes (from GVBD onwards) stained with Sgo2 antibody (red), CREST serum (green) and Hoechst (blue).

C) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards, corresponding to the time of anaphase I onset. PBE: Polar Body Extrusion.

D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively.

E) Quantification of D) showing Mps1 signal relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Mps1 signal averaged per oocyte (right dot plot) at the indicated time points. The number of kinetochore pairs (kinetochores in anaphase II) and oocytes analyzed is indicated. The scheme of measurements is shown on the right.

F) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst. Where indicated, oocytes were treated with Reversine from GVBD onwards, and treatment was renewed every 8 hours. Early CSF corresponds to 8 hours after GVBD (Reversine treated oocytes undergo anaphase I on average 4-5 hours after GVBD), and late CSF corresponds to 36 hours after GVBD (>29 hours in CSF arrest). PSCS: precocious sister chromatid segregation.

G) Quantification of total Sgo2 signal on spreads in early or late CSF arrest (16 hours or 30 hours after GVBD, respectively) relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Sgo2 signal averaged per oocyte (right dot plot) at the indicated stages. The number of kinetochore pairs and oocytes analyzed is indicated. The scheme of measurements is shown on the right. Meta: metaphase, ana: anaphase.

Data information:

On each graph mean is indicated, error bars are ± SD, asterisks indicate significant difference (**** = p<0,0001; ns = not significant) according to Mann-Whitney U test. AU: arbitrary units, n indicates number of analysed kinetochores, kinetochore pairs or oocytes, as indicated. Scale bars, 10μm. See also Fig EV2.
Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II (2021)
Figure 3

Figure 3.

Step-wise cohesin removal occurs independently of tension forces applied by a bipolar spindle.

(A-F): on the left, meiosis I, on the right, meiosis II.

(A and D) Oocytes in meiosis I (6 hours after GVBD) or meiosis II (left and right panels, respectively) were treated with STLC for 2.5 hours and fixed for whole mount immunofluorescence. Cold stable microtubules were stained with an antibody against tubulin (green), centromeres with CREST (red) and DNA with Hoechst (blue).

(B and E) Chromosome spreads at metaphase and anaphase of meiosis I (6 and 9 hours after GVBD) or meiosis II (left and right panels, respectively), in the presence or absence of STLC (for 2.5 hours) and Reversine (+Rev., for 1 hour) as indicated. Spreads were stained with CREST serum (red) and Hoechst (grey). Single chromosomes are present in anaphase I and single sister chromatids in anaphase II.

(C and F) Chromosome spreads exactly as described in B and E), except that spreads were additionally stained with an antibody against Rec8 (green). The CREST signal appears in red and DNA in blue. Rev: Reversine.

Data information:

Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed. See also Fig EV3.
Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II (2021)
Figure 4

Figure 4.

Oocytes without separase can be activated to undergo meiosis II and separate sister chromatids when rescued with wild-type separase from GV onwards.

A) Metaphase II oocytes from mice carrying a conditional, oocyte-specific invalidation of separase (Separaseflox/flox Zp3 Cre+, or Sep-/-) and oocytes from litter mates (Separaseflox/flox, or Sep+/+) were obtained after superovulation. In vivo matured control (Sep+/+) and Sep-/- oocytes are found in oviducts at comparable numbers, enclosed by cumulus cells. Example of cumulus enclosed oocytes from the oviduct is shown below.

B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated.

C) Same as B), except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards.

D) Metaphase-to-anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated, in GV. The schemes indicate the chromosome figures observed upon activation. Chromosome spreads are stained with CREST serum (green), and Rec8 antibody (red). DNA was stained with DAPI (blue).

Data information:

In (A), n indicates the number of mice and a mean number of oocytes per mouse of each genotype, error bars are + s.e.m. In (B and C) n indicates number of oocytes analyzed. In (D) n indicates the number of oocytes with the shown phenotype (e.g., upon successful activation) and the total number of oocytes analyzed. Scale bars indicate 5 µm in (B and C), and 10 µm in (D). See also Fig EV4A.
Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II (2021)
Figure 5

Figure 5.

Separase activity during meiosis I is required for centromeric cohesin removal in meiosis II.

A) Sep+/+ or Sep-/- oocytes were matured in vitro until metaphase II. Sep-/- oocytes were injected with wild-type separase or cleavage mutant separase encoding mRNAs in metaphase II. Oocytes were either fixed in metaphase II or activated and fixed in anaphase II. Spreads were stained with Rec8 antibody (red), CREST serum (green), and DAPI (blue) to label DNA.

B) Representative image of kinetochore individualization in Sep-/- oocytes injected with wild-type separase in metaphase II and fixed after activation, from A). Shown is staining with CREST serum (green), and DAPI (blue) to label DNA. Arrows indicate separated sister kinetochores. The scheme on the left illustrates the individualization of sister kinetochores (green arrows) at anaphase II.

Data information:

n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed Scale bars: 10 µm, except bottom panel in B), where the scale bar is 2 μm. See also Fig EV4B.
Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II (2021)
Figure 6

Figure 6.

Centromeric cohesin is not protected on bivalents with individualized kinetochores in meiosis II.

A) Live imaging movie of GFP-Sgo2 expressing oocytes (from GV onwards) undergoing meiosis I. The green channel to visualize GFP-Sgo2 localized to chromosomes is shown. Timepoints were taken every 10min, shown is a montage of selected frames such as indicated (hours: time after GVBD) at the metaphase-to-anaphase transition of meiosis I and in metaphase II. Each timepoint is comprised of an overlay of 11 z-sections of 3μm for GFP. The arrow heads indicate bivalents.

B) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP-Sgo2 expressing oocyte. Spreads were stained with Rec8 antibody (pink), CREST serum (green), Hoechst for DNA (blue), GFP-Sgo2 was visualized by GFP fluorescence (grey). The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms.

C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes.

D) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes.

E) Quantification of Rec8 signal found around and in between CREST signals that were close to each other. Below: scheme illustrating how Rec8 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines). Meta: metaphase, ana: anaphase.

Data information:

In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed, in D), the number of oocytes with the shown phenotype and the total number of oocytes analyzed. On each graph, mean is shown, error bars are +s.e.m. in C) and and ± SD in E), asterisks indicate significant difference (**** = p<0,0001) according to Mann-Whitney U test. AU: arbitrary units. The number of analysed oocytes in C) and of analyzed kinetochore pairs in E) is indicated. Scale bars: A) 50μm, (B-D) 10μm. See also Fig EV5 for additional timepoints and channels of the movie in A).
RIF1 and KAP1 differentially regulate the choice of inactive vs. active X chromosomes (2021)
Figure 1

Figure 1. Rif1 deficiency leads to female embryonic lethality at peri-implantation.

(A, B). Tables summarising the number and the sex of the pups recovered at weaning from Rif1+/- x Rif1+/- mice inter-crosses, either in a C57BL/6J (A) or in a mixed C57BL/6J-129/SvJ genetic background (B). The observed number of mice is compared to the expected number, based on the Mendelian ratio. p calculated by χ2.

(C). The table summarises the number and the sex of the embryos of the indicated genotypes, recovered from timed matings of Rif1+/- x Rif1+/- mice, in a C57BL/6J genetic background. The day of gestation (E) is indicated. (D). Representative images of Rif1-/- E7.5 embryos, female top and male bottom.
RIF1 and KAP1 differentially regulate the choice of inactive vs. active X chromosomes (2021)
Figure 2

Figure 2. Rif1 null female mESCs fail to upregulate Xist upon differentiation.

(A). Overview of the experimental design. Rif1+/+ and Rif1F/F mESCs were grown for two days in medium supplemented with 4-Hydroxytamoxifen (OHT) to induce the translocation into the nucleus of the Cre-recombinase, leading to Rif1 deletion in the Rif1F/F cells (Rif1-/-). The embryoid body (EB) differentiation protocol was then started to trigger XCI. OHT was kept in the medium during the first 24 hours of EB differentiation. Cells were differentiated up to 4 (RNA analysis) or 7 days (H3K27me3 IF).

(B). Representative western blot to monitor RIF1 levels after Cre-mediated Rif1 deletion and EB differentiation. SMC1: loading control.

(C). Time course analysis of Xist RNA expression by RT-qPCR during EB differentiation of Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) cells at the indicated timepoints. Rif1+/+ (solid line) and Rif1-/- (dashed line), female (black) and male (grey). Data are presented as mean ± standard deviation from three (female lines) or two (male lines) independent experiments. Statistical significance was determined using 2-way ANOVA comparing female Rif1+/+ to female Rif1-/- cell lines (****p ≤ 0.0001). Xist RT-primers Xist ex7 F and R were used. Values are normalised to a geometric mean consisting of the expression of Gapdh, Ubiquitin and β-Actin. (D). Bar plot summarising the number of cells showing H3K27me3-marked Xi as a percentage of total cells counted, in Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) female mESCs at the indicated days of EB differentiation. Averages ±standard deviation from three (day 4 and 7) and two (day 2) independent experiments (n>200).