EMBO/soda-nel huggingface dataset:
A spoonful of L-fucose - An efficient therapy for GFUS-CDG, a new glycosylation disorder (2021)
Figure 4

Figure 4. Biochemical characterization of GFUS deficiency in patient-derived fibroblasts

A Western blot analysis and quantification of GFUS. Expression of GFUS was analyzed by Western blotting on a 15% SDS PAGE with cytosolic fractions derived from control and patient fibroblasts. Quantification revealed a significant decrease of GFUS protein. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed.

B mRNA expression studies. qRT-PCR studies showed a significant decrease in mRNA level of GFUS by 40.0% (± 9.3%, **p = 0.0010) normalized to a control. Also, expression of SLC35C1 and SLC35C2 were significantly decreased in patient-derived fibroblasts. Data were obtained from fibroblasts; n = 10; experiment was independently repeated 3 times, for statistics an one-way ANOVA was performed.

C Conversion of GDP-D-mannose into GDP-L-fucose. The conversion GDP-D-[14C]mannose to GDP-L-[14C]fucose was analyzed in an in-vitro assay. After 1 h nearly half of GDP-D-mannose was transformed to GDP-fucose in the control cell line, whereas the patient´s fibroblasts were nearly unable to catalyze the reaction. Data were obtained from fibroblasts; n = 1.

D Lectin binding studies and quantification in fibroblasts. Aleuria aurantia lectin (AAL) was used to address the fucosylation level in fibroblasts. AAL staining was performed before and after treatment of the patient cell line with 100 µM L-fucose. Before sugar supplementation a significant loss of AAL signal strength was measured, whereas after treatment a significant re-fucosylation was detected. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 4 times, for statistics an unpaired t test was performed.

E Complementation study with patient-derived fibroblasts. Viral infection was used to introduce an empty cloning vector and the wild-type GFUS cDNA in patient cells, respectively. For analysis lysates of a control cell-line, a control cell-line supplemented with 100 µM fucose, patient cells, patient cells supplemented with 100 µM fucose, patient cells transfected with an empty vector and patient cells transfected with wild type GFUS were analyzed by AAL staining clearly indicating the disease-causing influence of the defective GFUS in the patient. Western blot analysis against GFUS further showed expression of wildtype-GFUS protein in the infected patient derived fibroblasts. Data were obtained from fibroblasts; n = 4; experiment was independently repeated 2 times, for statistics an one-way ANOVA was performed.

Data information: *p < 0.05; **p < 0.01; ***p<0.001. Bars and error bars represent mean ± SD. Exact p‑values are reported in the results part.
A spoonful of L-fucose - An efficient therapy for GFUS-CDG, a new glycosylation disorder (2021)
Figure 5

Figure 5. Aleuria aurantia lectin blot of the affected individual and controls and FACS analysis

A Aleuria aurantia lectin (AAL) was used to address the fucosylation level in sera of controls and the patient (left) which revealed significant loss of fucose residues in case of the patient. Data were obtained from serum; n = 6; experiment was independently repeated 3 times, for statistics an unpaired t test was performed.

B FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) before the start of fucose therapy.

C FACS analysis of PBMCs. Results of flow cytometry analysis of patient (red) and control (green) PBMCs stained with 1 µg/ml FAA lectin without (dark) or with 200 mM L-fucose (light) 8 weeks after therapy start

Data information: **p < 0.01. Bars and error bars represent mean ± SD. Exact p‑values are reported in the results part.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 1

Figure 1: The KATs Esa1 and Gcn5 counteract the KDAC Hos3 to promote the G1/S transition.

(A) Localization and function of the Hos3 deacetylase during mitotic division. Hos3 (in green) associates with the bud neck and with daughter-cell NPCs during nuclear migration into the bud. Hos3 delays the G1/S transition specifically in daughter cells through deacetylation of NPCs (Kumar et al, 2018).

(B) Growth inhibition upon overexpression of HOS3-NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1. 10-fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids, were spotted onto SC-Glu and SC-Gal medium (to activate the GAL1 promoter) and incubated at 25 °C for 3 days. Note that HOS3-NLS (under the control of the native HOS3 promoter) does not affect cell growth.

(C) esa1-ts and gcn5Δ esa1-ts mutants have bud emergence defects. (Top) Cells of the indicated strains were arrested in G1 by treatment with ɑ-factor for 2.5 h at 25 ºC, shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC. Cells were fixed at the indicated times and the presence of buds was assessed by microscopy. Mean and SEM are derived from n = number of independent experiments. At least 200 cells were scored for each strain and time point. (Bottom) Bright field images of the indicated strains 60 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.

(D) Inactivation of ESA1 and GCN5 delays DNA replication. Cells of the indicated genotypes were synchronised as in panel C, and DNA content was evaluated by flow cytometry. Numbers indicate time in minutes after the release. This experiment was repeated three times with similar results; one experiment is shown.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 2

Figure 2: Acetyl-mimic Nup60 partially rescues the Start defects of esa1-ts cells.

(A) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60. (Top) Nup60-GFP was immunoprecipitated from extracts of the indicated strains, and its acetylation state probed with anti-AcLys antibodies. (Bottom) Total extracts probed with anti-HA antibodies to verify KAT overexpression.

(B) nup60-KN partially rescues the budding defect of esa1-ts cells. (Left) Budding of cells of the indicated strains were determined as in Figure 1C. At least 200 cells were scored for each strain and time point. Data from 3 independent experiments is represented as mean and SEM (esa1-ts, esa1-ts nup60-KN). (Right) Bright field images of the indicated strains 40 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.

(C) mRNA levels of CLN2, CDC21, SVS1, and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature, with samples collected at indicated times. Data from 3 independent experiments is represented as mean and SEM.

(D) nup60-KN mutation partially rescues the delay in synthesis of the G1/S cyclin Cln2 in esa1-ts cells. Cells of the indicated strains were processed as in (B) and the amount of Cln2-HA protein at the indicated times was assessed by western blot. % of budded cells is indicated below for each corresponding strain and time point. Note the slow, inefficient budding in esa1-ts mutants. In WT, the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle, which is absent in esa1-ts cells.

(E) nup60-KN partially rescues the Whi5 export defect of esa1-ts daughter cells. Composite of bright field and Whi5-mCherry (left) and quantification of Whi5 nuclear export (right) in mother (M) and daughter (D) cells of the indicated strains. Whi5 export (arrows) is delayed in esa1-ts mothers and daughters compared to WT (p < 000.1, Log-rank Mantel-Cox test); esa1-ts nup60-KN advances Whi5 export relative to esa1-ts in daughters (p=0.0105), but not in mothers (p > 0.05). 3 z-confocal slices spaced 0.5 µm were acquired every 3 min; maximum projections of selected timepoints are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. n = number of cells, pooled from two independent experiments with similar results.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 3

Figure 3: Esa1 and Nup60-KN promote the nuclear export of CLN2 mRNA.

(A) Composite of bright field and Whi5-tdTomato (BF/Whi5) and CLN2-PP7 mRNA labelled with PCP-NLS-GFP (CLN2 mRNA) in mother (M) and daughter (D) cells of the indicated strains. Arrows indicate nuclear foci. Brighter GFP nucleoplasmic areas may correspond to the nucleolus. Insets show enhanced-contrast images to visualise cytoplasmic mRNA particles (arrowheads). Numbers indicate minutes relative to Whi5 nuclear import. Maximum projections of whole-cell Z-stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1-ts images, where single Z-slices are shown for clarity. Scale bar: 4 µm (inset: 1 µm).

(B) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 import. Symbols represent individual values; lines were generated by smoothing of the nearest 3 neighbouring values (0th order polynomial).

(C) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 export.

(D) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export, normalised relative to the nuclear background.

(E) Number of cytoplasmic CLN2-PP7 mRNA foci in early G1 (5 minutes after Whi5 import) and at the G1/S transition (5 min after Whi5 export).

(F) The position of nuclear CLN2-PP7 foci (marked with arrowheads in the examples) was scored relatively to the nuclear periphery (visualised with PCP-NLS-GFP) in the indicated strains. The fraction of non-perinuclear CLN2-PP7 foci is significantly increased in esa1-ts cells compared to the WT and rescued by nup60-KN mutation (two-sided Fisher's exact test p = 0.0209 and p = 0.0227). Scale bar, 1 µm.

Data information: In (D-E), boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (D): WT vs. esa1-ts p < 0.0001, WT vs. esa1-ts nup60-KN p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.9845; in (E): WT vs. esa1-ts p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.002. Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 4

Figure 4: Nuclear export of mRNA is inhibited by the KDAC Hos3, and promoted by the KAT Esa1 and Nup60 acetylation.

(A) Illustration of the physical interactions (dashed lines) of the NPC with mRNA export factors.

(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.

(C) A high-copy plasmid containing SAC3 rescues the lethality of Hos3-NLS overexpression. Strains carrying a high copy plasmid containing the SAC3 ORF (nucleotides 467-3905), or the empty vector, were grown as in (B).

(D) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions.

(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions as in (D).

(F) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed as in (E).

Data information: In (D-F), arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 5

Figure 5: Nup60 deacetylation in daughter cells displaces Sac3 from NPCs and delays Start.

(A) Depletion of Hos3 or expression of acetyl-mimic Nup60 (nup60-KN) increases the nuclear localisation of Sac3 in G1. Cells of the indicated strains were imaged by time-lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 (after cytokinesis). The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels. Fluorescence intensity was measured in sum projections of whole-cell Z-stacks, by segmentation of either the nuclear area in the mCherry channel, or of the whole cell in the brightfield channel. The ratio of Sac3/Nup49 intensities was then normalised relative to the mean intensity of wild-type mothers.

(B) Inactivation of Esa1 decreases Sac3 nuclear levels. Wild-type (WT) and esa1-ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC, shifted to 37 ºC, released from the mitosis block in fresh medium at 37 ºC, and imaged by time-lapse microscopy. Fluorescence levels were quantified in G1 as in (A).

(C) Rapamycin-dependent dimerisation abolishes Sac3 mother/daughter asymmetries. NUP60-mCherry-FKBP SAC3-GFP-FRB cells were incubated with rapamycin (RAPA) to trigger FRB-FKBP heterodimerization, or with DMSO as control. Fluorescence levels were quantified in G1 cells as in (A), 15 to 30 minutes after addition of the drug.

(D) Sac3 anchoring to the nuclear basket advances Start in esa1-ts daughter cells. Composite of bright field and Whi5-mGFP (top) and quantification of Whi5 nuclear export timing (bottom) in wild-type (WT) and esa1-ts mother (M) and daughter (D) cells treated with either rapamycin (RAPA) or DMSO and expressing Nup60-FRB and Sac3-mCherry-FKBP. Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells (DMSO vs RAPA, p > 0.05, Log-rank Mantel-Cox test), but it advances Whi5 export in esa1-ts daughters (p = 0.0001). Whi5 export efficiency was slightly improved also in mother cells (p = 0.0374). 8 z-confocal slices spaced 0.4 µm were acquired every 3 min; maximum projections are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm.

Data information: In (A-C), arrowheads point to daughter cells, and in (D), to Whi5 export. In (A-C), boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 6

Figure 6: Daughter-cell specific Nup60 deacetylation inhibits GAL1 expression.

(A) Time Lapse microscopy of WT and nup60-KN cells expressing GAL1pr:sfGFP and Nup60-mCherry at the indicated times of galactose induction. Scale bar, 4 µm.

(B) Depletion of Hos3, and expression of acetyl-mimic Nup60 (nup60-KN) enhances GAL1 expression. WT, hos3Δ, nup60-KN and nup60-KN hos3Δ cells were shifted to galactose and imaged by time-lapse microscopy to monitor GAL1pr:sfGFP expression during 7 hours. Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60-mCherry was quantified from sum projections of whole-cell Z-stacks at the indicated times. At least 200 cells were scored for each strain and time point. Shaded areas indicate the SEM.

(C) mRNA levels of GAL1 were determined for wild type (WT) and nup60-KN cells at the indicated times after galactose addition. One of two independent experiments with similar results is shown (mean and SEM from 3 technical replicates).

(D) The GFP intensity of mother/daughter pairs for cells in B, at 5-minute intervals after galactose addition (left) and 425 min after galactose addition (right). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.001; ns, p > 0.05, two-tailed paired t-test. n = number of cells. One of two independent experiments with similar results is shown.

(E) Expression of GAL1pr:sfGFP induced with galactose (left) or ß-estradiol (right), in the presence of the ß-estradiol-dependent transactivator Gal4-ER-VP16. Smooth lines show GFP fluorescence intensity (left x axis); lines with circles show the difference in GFP intensity between the indicated strains (right x axis). The difference between wild type and hos3∆, and between wild type and nup60-KN, increases continuously over time in response to galactose, but not to ß-estradiol.
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast (2022)
Figure 7

Figure 7: Sac3 anchoring to Nup60 promotes GAL1 expression.

(A) Cells of the indicated strains were incubated with galactose in the presence of either rapamycin (RAPA) to induce FRB-FKBP heterodimerization, or DMSO as control. GAL1pr:sfGFP expression was monitored over time as in Figure 6B.

(B) (Top) Representative images of the indicated cells in rapamycin or DMSO, 250 min after galactose addition. Scale bar 4 µm. (Bottom) Mother/daughter pairs were quantified as in Figure 6D at 250 min after galactose addition (pink shaded area in A). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. **, p ≤ 0.01; *, p ≤ 0.05; ns, p > 0.05, two-tailed paired t-test for M-D comparisons, unpaired for comparisons between strains. n = number of cells. One of two independent experiments with similar results is shown.
Genome‐wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development (2014)
Figure 1

Genome‐wide RNAi screen for gene inactivations that suppress the accumulation of SQST‐1::GFP aggregates in rpl‐43 mutants

A–D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.
E Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.
F Mapping and cloning of bp399. LG II: linkage group II.
G Protein sequence of RPL‐43. The mutated residue in bp399 is highlighted in red.
H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.
J–Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.
R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.
S Functional classification of the identified genes. The number of genes in each functional class is shown in parentheses.
Genome‐wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development (2014)
Figure 2

Loss of function of a variety of signaling pathways suppresses the rpl‐43 phenotype

A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.
C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.
D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.
E Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.
F mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.
G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.
I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.
K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.
L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.
M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.
O, P SQST‐1::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.

Data information: 200 young adult animals for each genotype were collected for analysis in (D), (E), and (K). Scale bars: 20 μm (A, B, G–J, M–P). (A), (G), (I), (M), and (O): DIC images of the animals in (B), (H), (J), (N), and (P), respectively.
Genome‐wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development (2014)
Figure 3

XBP‐1‐mediated ER stress suppresses the rpl‐43 phenotype

A, B Phsp‐4::GFP is very weakly expressed in wild‐type animals.
C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.
D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals.
F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.
H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.
J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.
L Functional interaction of ER stress‐induced genes.
M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.

Data information: (A), (D), (F), (H), and (J): DIC images of the animals in (B), (E), (G), (I), and (K), respectively. Scale bars: 100 μm (A, B, D–G); 20 μm (H–K).