EMBO/soda-nel huggingface dataset:
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 3

Figure 3 - Bak is shifted into the cytosol in parallel to Bax by retrotranslocation.

A Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Fig. E2a).

B Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from 3 independent experiments with n ≥ 100.

C Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD.

D Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 µM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Fig. E3C, D). Akt1 and Tom20 serve as fractionation controls. n = 3.
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 4

Figure 4 - The C-terminal membrane anchor determines the localization of Bax and Bak.

A The influence of the C-terminal membrane anchor (MA) on the differential localization and function of Bax and Bak has been analyzed using the wild-type proteins of Bax (blue) and Bak (green) and MA substitutions of both proteins resulting in the chimeras BaxTBak and BakTBax.

B Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from 7 independent experiments with n ≥ 100 cells.

C Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale 10 µm. n ≥ 5.

D Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 5

Figure 5 - Mitochondrial Bax is activated apoptosis stimulus-independent.

A Caspase 3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA are displayed.

B Staurosporine (STS, 1 µM)-induced caspase 3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. p-values according to One Way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (p < 0.001).

C Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior or after treatment with 1 µM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.

D HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 µM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.

E Flow cytometry analysis of Annexin V-staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS-treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.

F Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 µM) was added for 24 hrs before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.

G Quantification of colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment (Fig. 5F). Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA. BaxTBak expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).

H Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. p-values according to One Way ANOVA.
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 6

Figure 6 - Bax and Bak are regulated by the same mechanism.

A FLIP of BaxTBakSS without (○, broken line) or with (●, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements.

B Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as control for PARP cleavage. Similar sample loading is controlled by actin. n = 4.

C Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3.

D Caspase 3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as control. Data represent averages ± SEM. n = 4. p-values according to One Way ANOVA using the Holm-Sidak method are displayed.

E Comparison of Bax and Bak localization and activity dependent on the MA (Bax MA, blue rectangle) using wild-type Bax (i, blue), Bax S184V (ii), BakTBax (iv) and BakTBax S184V (iii). Direct comparison of Bax S184V (ii) and BakTBax S184V (iii) reveals MA-independent influences of Bax and Bak on protein localization and activity (I). The impact of the S184V substitution (*) on translocation, retrotranslocation and activity is shown by differences between BakTBax and BakTBax S184V (II). The additional effect of Bax MA binding to the hydrophobic groove of Bax (III) is indicated by the comparison of both Bax variants (i+ii) versus both Bak variants (iii+iv).

F STS-induced caspase 3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.

G Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as control for PARP cleavage and actin is the loading control. n = 3.

H Plot of Bax (●) and Bak (○) retrotranslocation rates vs. hydrophobicity of the MA of the constructs. R2 = 0.95.
Differential retrotranslocation of mitochondrial Bax and Bak. (2015)
Figure 7

Figure 7 - Bax activation does not per se depend on mitochondrial Bax levels.

A Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.

B Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.

C Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.

D HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.

E Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3.

F PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as loading control. n = 3.

G Bax (blue, left) and Bak (green, right) are retrotranslocated similarly by pro-survival Bcl-2 proteins. The shuttling shifts also OMM-integral Bax and Bak into the cytosol dependent on the interactions between hydrophobic Bcl-xL groove and BH3 motif of either Bax or Bak. While Mcl1 shuttles Bax and Bak like Bcl-xL, Bcl-2 only accelerates Bax retrotranslocation. Fast shuttling from the mitochondria into the cytosol at the level of Bax retrotranslocation in proliferating cells shifts Bax and Bak into the cytosol and inhibits their pro-apoptotic activities in parallel (top). Wild-type Bak is usually shuttled at low retrotranslocation rates and thus accumulates on the mitochondria (bottom, right). Shuttling at similar speed also causes mitochondrial Bax accumulation but results in stimulus-independent Bax activation (bottom, left). Therefore cells are required to shuttle Bax at high rates to prevent commitment to apoptosis.
Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 1

Figure 1. Cdc14 is required for intra-chromosomal DNA repair by HR.

A) Ten-fold serial dilutions from overnight cultures of wild-type and cdc14-1 cells dropped and grown on solid rich media or media containing MMS at 25, 28, 30 or 33ºC. Note that cdc14-1 cells exhibit growth sensitivity to MMS at 28ºC and 30ºC compared to wild-type cells.

B) Ten-fold serial dilution from mid-log phase cultures of wild-type and cdc14-1 cells grown on solid rich media or media containing mock DMSO (as non-treated control), 4NQO, HU, phleomycin and benomyl at 30ºC. Note that cdc14-1 cells present a market sensitivity to all DNA damaged agents tested.

C) Left panel: Schematic representation showing relevant genomic structure of the strain used to assess intra-chromosomal repair. The location of a MAT specific probe and the restriction endonuclease cleavage sites used for Southern blot analysis to detect repair product formation are indicated. Arrow depicts the localization of the double-strand break. Right panel: Physical analysis of intra-chromosomal repair in wild-type and cdc14-1 cultures at the semipermisive temperature. After DSB formation by the expression of the HO, glucose was added to repress it, thus allowing repair with HM donor sequences. Genomic DNA was digested with StyI, separated on agarose gel and blotted. Blots were hybridized with a probe corresponding to the MATa-distal sequence. A second probe for the actin gene was used to control the amount of genomic DNA loaded at each time-point. Immunoblot analysis of Rad53 during mating type switching experiments is shown. Coomassie blue staining is depicted as loading control. Graphs show quantification of gene conversion (GC) leading to the re-establishment of MATa or switching to MAT and DSB formation. The data was normalized with the actin loading control. Graphs show the mean ± S.D. from three independent experiments. Replicates were averaged and statistical significance of differences assessed by a 2-tailed unpaired Student t test.

Data information: MMS, methyl-methanesulfonate. DMSO, dimethyl sulfoxide. 4NQO, 4-nitroquinoline-1-oxide. HU, hydroxyurea. Raf, raffinose. Gal, galactose. Glu, glucose. DSB, Double strand break. GC, gene conversion.
Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 5

Figure 5. The spindle pole body component Spc110 is a target of Cdc14 during the DNA damage response.

A) Identification of Cdc14 phospho-targets during the DNA damage response by mass spectrometry analysis. Wild-type and cdc14-1 cells were grown overnight and blocked in G2/M by using nocodazole to avoid cell cycle-dependent changes in protein phosphorylation between both strains. After the block was attained, cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h. Differential phosphorylation of phospho-peptides detected between the wild-type and cdc14-1 were grouped into broad categories depending on the molecular function of the proteins. The table includes the DNA damage and checkpoint-related proteins with Cdc14-dependent hyper-phosphorylated status and the relative ratio between the wild-type and cdc14-1 during the DNA damage response. Red and blue indicate relative amount of the residue phosphorylation between both strains (Red, high; Blue, low).

B) Cdc14 dephosphorylates Spc110 in response to a DSB. Spc110 was tagged with the 6HA epitope in wild-type, cdc14-1 and spc110S36-91A backgrounds. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as loading control.

C) Lambda-phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response. Cells were grown overnight in raffinose containing media (line 1) and incubated with galactose for 2.5 hours to induce HO expression (line 2). Extracts from raffinose-containing cultures were treated with mock (line 3) or λ-PPase (line 4). All samples were separated in a Phos-Tag containing gel and blotted. Coomassie staining is shown as loading control.

D) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response. Spc110 was labelled with the 3HA tag in both wild-type and esp1-1 background strains. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as a control for loading.

Data information: Raf, raffinose. Gal, galactose. λ-PPase, Lambda phosphatase. Asterisk denotes coomassie blue staining as loading control for Western blots.
Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 6

Figure 6. Cdc14 activity is needed for stabilization of the metaphase spindle in response to DNA damage.

A) Time-lapse experiments of living cells harbouring a non-reparable DSB were carried out after expressing the HO endonuclease in wild-type and cdc14-1 mutant cells. Spc110-RFP was used as SPB reporter and Tub1-GFP was used as tubulin marker. Three z-planes images every 5-second intervals over a period of 5 minutes were captured and used to quantify the average SPB track length and velocity using Spc110-RFP as SPB marker. At least 100 cells were scored. A Western blot is shown to confirm the equal amount of Tub1-GFP in both wild-type and cdc14-1 backgrounds. Coomassie blue staining is shown as loading control. A representative maximum projection image is depicted. Scale bar: 3μm.

B) The steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to DNA damage. Wild-type, cdc14-1 and spc110S36-91A cells expressing Spc110-RFP were scored for their ability to maintain spindle polarity at the axial plane of the cell upon induction of a single DSB induced by the HO endonuclease. A maximum projection of nine z-planes was used to score the cells. At least 100 cells from three independent experiments were scored. Graph represents the percentage ± SD of cells with oriented/miss-oriented SPBs with respect to the axial plane of the cell. P value was calculated using a 2-tailed unpaired Student t test.

C) The asymmetric loading of dynein into astral MTs emanating from the daughter SPB is affected in cdc14-1 and spc110S36-91A cells during the DNA damage response. Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1-GFP in strains containing Spc110-RFP. Graph represents the percentage ± SD of cells grouped into three categories depending on the pattern of dynein distribution on astral MTs: Type1, Dynein is exclusively loaded on astral MTs emerging from the daughter SPB; Type 2, Dynein is equally loaded on astral MTs emanating from both SPBs; Type 3, Dynein is absent from astral MTs. At least 100 cells from three independent experiments were scored. A representative picture showing the three patterns is included. Scale bar: 3μm.

Data information: DSB, Double Strand Break. SPB, Spindle Pole Body.
Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 7

Figure 7. Microtubule destabilization affects DSB-SPB interaction and DSB repair by homologous recombination.

A) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm.

B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the -factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. FACS analyses are shown. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test.

Data information: DSB, double-strand break. SPB, Spindle Pole Body. DMSO, dimethyl sulfoxide. Nz, nocodazole. GC, gene conversion. Raf, raffinose. Gal, galactose. Glu, glucose.
Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair (2016)
Figure 8

Figure 8. Cdk-dependent phosphorylation of Spc110 is essential to stabilize the metaphase spindle, stimulate DSB-SPB interaction and promote DNA repair by homologous recombination.

A) Growth sensitivity to MMS and phleomycin of a Cdk phospho-deficient mutant of Spc110. Ten-fold serial dilutions from overnight cultures of wild-type, spc110S36-91A and a cdc14-1 cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.

B) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Spc110 and spc110S36-91A were labelled with the RFP in a background containing Ddc2-GFP as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.

C) Time-lapse experiments of living cells expressing the HO endonuclease in wild-type and spc110S36-91A mutants cells. Five z-planes every 5-second intervals over a period of 5 minutes were captured. Quantifications were made using the average SPB track length and velocity of at least 100 cells. Spc110 and spc110S36-91A were labelled with the RFP and used as SPB reporter.

D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. FACS profiles for DNA content are included. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.

Data information: DMSO, dimethyl sulfoxide. MMS, methyl-methanesulfonate. SPB, Spindle Pole Body. DSB, double-strand break. Raf, raffinose. Gal, galactose. GC, gene conversion. CO, crossover.