EMBO/soda-nel huggingface dataset:
A Role for Macro-ER-Phagy in ER Quality Control. (2015)
Fig 1

The general Atgs Atg1 and Atg8 and the selective Atg Atg11, but not other selective Atgs, are required for macro-ER-phagy.

A. GFP-Snc1-PEM protein accumulates in atg1∆, atg8∆, and atg11∆ mutant cells. Lysates were prepared from wild type (WT), ypt1-1 (for comparison), atg1∆, atg8∆, and atg11∆ mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control. B. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1∆, atg8∆, and atg11∆ mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61. C. UPR is induced in atg1∆, atg8∆, and atg11∆ mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response. D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19∆, atg11∆, atg32∆, and atg36∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.
A Role for Macro-ER-Phagy in ER Quality Control. (2015)
Fig 2

Depletion of Atg9 and Atg2, but not Atg18, result in a macro-ER-phagy phenotype different from that of other core Atgs, and <italic>atg9∆</italic> is epistatic to <italic>atg11∆</italic>.

A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A–1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B), and induction of the UPR response (C,atg1∆ is shown as a positive control). B. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. D-G. Atg9 is epistatic to Atg11 in macro-ER-phagy. ATG9 was deleted in wild type and atg11∆ mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in Fig 1 legend. D. Deletion of ATG9 in wild type (WT) or atg11∆ mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in atg11∆ mutant cells. E. Deletion of ATG9 in wild type or atg11∆ mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the atg9∆ single-, and atg9∆ atg11∆ double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of atg11∆ mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain). F. UPR is induced in atg11∆, but not in atg9∆ single- and atg9∆ atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM. G. UPR can be induced in atg9∆ single-, and atg9 atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.
A Role for Macro-ER-Phagy in ER Quality Control. (2015)
Fig 3

Atg9 is epistatic to Ypt1 in macro-ER-phagy and not to the ER-exit regulator Sec12, which is not defective in this process.

A-C.ATG9 was deleted in ypt1-1, WT and sec12ts mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed as described for Fig 1A–1C, respectively. A. Snc1-PEM is increased ~3 fold in atg9∆ and sec12ts as compared to ~20 fold in ypt1-1 mutant cells. Importantly, atg9∆ is epstatic to the ypt1-1, but not to the sec12ts, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data. B. Whereas deletion of ATG9 in ypt1-1 mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in sec12ts mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. C. Deletion of ATG9 in ypt1-1 mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in sec12ts mutant cells (p-value = 0.05). D. Atg9 is present on aberrant ER structures that accumulate in ypt1-1 mutant cells. WT and ypt1-1 mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the ypt1-1 mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed). E. GFP-Snc1-PEM is delivered to the vacuole for degradation in sec12ts mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of sec12ts mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.
A Role for Macro-ER-Phagy in ER Quality Control. (2015)
Fig 4

GFP-Snc1-PEM accumulates in APs of <italic>vps21∆</italic> mutant cells and macro-ER-phagy is independent of UPR induction.

A. The ypt1-1 mutation is epistatic to vps21∆ in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, vps21∆, ypt1-1 and ypt1-1 vps21∆ double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of vps21∆ mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the vps21∆ mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% ypt1-1 and ypt1-1 vps21∆ mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize. B-D. UPR induction is not required for macro-ER-phagy. The UPR regulators Ire1 or Hac1 were deleted in the WT and ypt1-1 mutant cells. The following effects of overexpression of GFP-Snc1-PEM in WT and ypt1-1 mutant cells, without and with ire1∆ or hac1∆, were analyzed as described for Fig 1A–1C, respectively: protein level (B), accumulation of GFP-Snc1-PEM in aberrant structures (C), and UPR induction (D). B. Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or ypt1-1 mutant cells. C. Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1-1 mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures. D. Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1-1 mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.
A Role for Macro-ER-Phagy in ER Quality Control. (2015)
Fig 5

Snq2-yEGFP is another macro-ER-phagy cargo and its co-overexpression with DsRed-Snc1-PEM has a synergistic effect.

A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A–1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B), and induction of the UPR response (C, GFP-Snc1-PEM is shown for comparison). B. The ER marker Sec61 was tagged with mCherry at its C-terminus in WT and ypt1-1 mutant cells. Shown from left to right: DIC, GFP, mCherry, Merge, % cells with intracellular Snq2 and % cells in which Snq2 co-localized with Sec61. D-F. Synergistic effect of co-overexpression of Snq2-GFP with DsRed-Snc1-PEM in atg11∆ mutant cells. WT (left) and atg11∆ mutant cells (right) were transformed with plasmids for overexpression of Snq2, Snc1-PEM or both Snq2 and Snc1-PEM. D. Immunoblot analysis and quantification. Shown from top to bottom: Snq2-yEGFP (using anti-GFP antibodies), Ds-Red-Snc1-PEM (using anti-Snc1 antibodies), G6PDH (loading control), and a bar graph showing fold increase of Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) in atg11∆ mutant cells over WT. E. Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig). F. Fluorescence level of intracellular structures (ratio over PM) in atg11∆ mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in atg11∆ mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.
A Role for Macro-ER-Phagy in ER Quality Control. (2015)
Fig 7

Live-cell microscopy analysis of ER-resident proteins upon overexpression of GFP-Snc1-PEM.

A. In cells defective in vacuolar proteolysis (pep4∆ prb1∆), Hmg1 co-localizes with 70% of intra-cellular Snc1-PEM, but Sec13 does not (at least 36 cells were analyzed for each strain). No co-localization was observed for resident ER proteins and overexpressed Snc1-PEM in WT cells (PEP4 PRB1). Hmg1 (left) and Sec13 (right) were tagged with mCherry at their C-terminus in PEP4 PRB1 (WT) and pepb4∆ prb1∆ mutant cells. Cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM and analyzed by live-cell microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge (yellow), % cells with intra-cellular Snc1-PEM, and % co-localization of the ER protein with intra-cellular Snc1-PEM. B. The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of PEP4 PRB1 (WT) and pepb4∆ prb1∆ mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% pep4∆ prb1∆ mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain). C. >80% of ypt1-1 mutant cells, regardless if they are PEP4 PRB1 or pep4∆ prb1∆, accumulate aberrant GFP-Snc1-PEM structures. Hmg1 co-localizes with >70% of these structures, whereas Sec13 co-localizes with only ~15% of these structures (36–43 cells were analyzed for each strain). The experiment was done as described for Panel A. D. Summary of microscopy analysis of ER-resident proteins co-localization with GFP-Snc1-PEM in cells overexpressing GFP-Snc1-PEM: WT (PEP4 PRB1) vs pep4∆ prb1∆ (left), and ypt1-1 PEP4 PRB1 vs ypt1-1 pep4∆ prb1∆ (right). Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig. In YPT1 cells, whereas >60% of Hmg1 and Sec61 (green) co-localize with Snc1-PEM in strains defective in vacuolar proteolysis, Sec12, Sec13 and Kar2 are not changed significantly (blue and grey). Hmg1 and Sec61 (green) co-localize with Snc1-PEM in >70% of ypt1-1 and >80% of ypt1-1 pep4∆ prb1∆ mutant cells, whereas Sec13 and Kar2 (blue and grey) do so in <15% of the cells. The level of Sec12 co-localization with Snc1-PEM is intermediate, ~38% in ypt1-1 and ypt1-1 pep4∆ prb1∆. Error bars represent STDEV. Results in panels A and B represent at least two independent experiments.
Liver-directed gene therapy for ornithine aminotransferase deficiency. (2022)
Figure 1

Fig. 1. Improvement of retinal disease of Oatrhg mice by liver-directed gene transfer.

(a) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV-OAT at the doses of 1x1013 (n=9) and 3x1013 genome copies (gc)/Kg (n=6), or AAV-GFP at the dose of 1x1013 gc/Kg (n=9). Normal range was established on wild-type mice (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVA test).

(b) Amplitude of a- and b-waves under scotopic and photopic conditions (mean ± SEM) in uninjected wild-type (WT) mice (blue circles, n=10), Oatrhg mice injected with AVV-OAT of 1x1013 gc/kg (red circles, n=12) or 3x1013 (gray circles, n=6) and Oatrhg mice injected with AVV-GFP (black circles, n=8) at 11 months of age (9-months post-injection). Two-way ANOVA test; *p <0.05; **p<0.01.

(c) Plasma ornithine and lysine concentrations at 12 months of age (10-months post-injection) in Oatrhg mice injected with AAV-OAT (n=3) or AAV-GFP (n=3); WT mouse controls (n=5) are also shown; each sample was analyzed as triplicates, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.

(d) Ornithine concentrations in the eye cups of Oatrhg mice at 12 months of age (10-months post-injection). Data are shown as Z-scores; one-way ANOVA with False Discovery Rate correction was used for comparison between groups. Means ± SEM are shown; *p <0.05 ***p<0.001, ****p<0.0001; ns not statistically significant difference.

(e) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV-GFP or AAV-OAT. A WT mouse liver was included as control. Vinculin was used as loading control.

(f) OAT activity on liver lysates of Oatrhg mice injected with AAV-OAT or AAV-GFP vectors (n=4 for each group). Data are shown as means ± SEM. Unpaired T-test; *p<0.05.

(g) Liver immunohistochemistry showing approximately 10% of OAT-positive cells in Oatrhg mice injected with AAV-OAT. Livers of WT and Oatrhg mice injected with AAV-GFP are shown as controls. 5x (images on the left side) and 20x (images on the right side) fields of view are shown.

(h) Representative H&E staining of the retinas of 11-month-old Oatrhg mice injected with AAV-GFP or AVV-OAT at the doses of 1x1013 or 3x1013 gc/Kg; age-matched WT mice and uninjected Oatrhg mice were included as controls (40X). Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.
Liver-directed gene therapy for ornithine aminotransferase deficiency. (2022)
Figure 2

Fig. 2. Ultrastructure of photoreceptor outer segment and retinal pigmented epithelium of Oatrhg mice after liver-directed gene transfer of OAT.

(a) Representative electron micrographs of the mouse retina of an AAV-GFP injected Oatrhg mouse showing retinal pigmented epithelium (RPE) and adjacent abnormal outer segment (OS) disk membranes (black arrows). Phagolysosomes containing undigested OS disc membranes are present (asterisks).

(b) Retina of AAV-OAT injected Oatrhg mice showing photoreceptor disk membranes (white arrows) and RPE ultrastructure comparable to age matched WT mice (c, white arrows).

(c) Control retina of WT mouse is shown as comparison.
Liver-directed gene therapy for ornithine aminotransferase deficiency. (2022)
Figure 3

Fig. 3. Improvement phenotype in OatΔ mice following AAV-mediated liver-directed OAT gene transfer.

(a) Plasma ornithine concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) OatΔ mice. Normal range of wild-type mice is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.

(b) ERG at six months post-injection in OatΔ mice injected with either AAV-GFP (n=6) or AVV-OAT (n=5); wild-type (WT) controls are also shown (n=5). Two-way ANOVA; test *p <0.05; **p<0.01; ***p<0.001. Data are shown as averages ± SEM.

(c) Retinal H&E (40X) of OatΔ mice injected with either AAV-GFP or AAV-OAT. Representative of three mice per groups are shown; WT mice were included as control. Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.

EXPANDED VIEW FIGURES
The MMS22L-TONSL heterodimer directly promotes RAD51- dependent recombination upon replication stress (2016)
Figure 1

Figure 1. MMS22L foci form during S phase of the cell cycle and co-localize with HR proteins.

A Immunofluorescence images of MMS22L foci formation at etoposide (ETP)-induced DSBs during S, M and G1 phases of the cell cycle in HeLa cells. Scale bar: 5 μm.

B Quantification of a representative experiment from (A) shows number of MMS22L foci per nucleus during indicated cell cycle phases (n = 2; nnuclei ≥ 60).

C Immunofluorescence images of CPT-induced MMS22L, RPA2, BRCA2 and RAD51 foci in U2OS cells. Scale bar: 5 μm.

D Quantification of a representative experiment (n = 3) from (C) shows percentage of γH2AX, RPA2, BRCA2 and RAD51 foci co-localizing with MMS22L foci (nnuclei = 30; nuclei with ≥ 25 MMS22L foci were analyzed).

(B, D) Boxplots represent distributions per nucleus; boxes indicate the 25–75 percentile and whiskers the 10–90 percentile. Horizontal lines mark the medians. Statistical analysis: Mann–Whitney U test; *** P ≤ 0.0001; ns, not significant.
The MMS22L-TONSL heterodimer directly promotes RAD51- dependent recombination upon replication stress (2016)
Figure 2

Figure 2. MMS22L-TONSL directly interacts with RPA-coated ssDNA.

A Electrophoretic mobility shift assay monitoring MMS22L-TONSL binding to ssDNA without RPA and with RPA-coated ssDNA.

B Quantification of data as in (A) shows percent MMS22L-TONSL binding to ssDNA with or without RPA, n = 2; error bars, SEM.

C Schematic (left) and western blots (right) of streptavidin and NiNTA pulldowns of his-tagged MMS22L-TONSL, RPA and biotinylated ssDNA.

D Yeast two-hybrid analysis shows the effect of deletion of the amino acids 384-522 of MMS22L on the interaction with RPA. The interaction between indicated MMS22L variants fused to LexA DNA-binding domain and RPA1 fused to GAL4 activating domain (GAD) is assessed by survival on media containing 3-aminotriazole (3-AT) in the absence of histidine and by β-galactosidase activity (X-Gal) assays.

E Images of MMS22L foci formed in U2OS cells stably expressing wild-type (WT) or Δ384-522 variants of siRNA-resistant HSS-MMS22L, depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours. Scale bar: 5 μm.
The MMS22L-TONSL heterodimer directly promotes RAD51- dependent recombination upon replication stress (2016)
Figure 3

Figure 3. MMS22L-TONSL localizes to active and stalled replication forks.

(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.

A Abundance of MMS22L-TONSL at active and HU-stalled replication forks, and fully replicated DNA.

B, C MMS22L dynamics at stalled replication forks. Quantification shows average relative abundance of indicated proteins detected in immunoblots, n = 3 except of time-point 5 h, n = 1; error bars, SEM.

D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.
The MMS22L-TONSL heterodimer directly promotes RAD51- dependent recombination upon replication stress (2016)
Figure 4

Figure 4. MMS22L is required to promote reversal of uncoupled replication forks.

A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.

B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25–75 percentile and whiskers the 10–90 percentile; horizontal lines mark the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann–Whitney U test; *** P ≤ 0.0001.

C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.

E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.

F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann–Whitney U test; ** P ≤ 0.01, n = 2.