EMBO/soda-nel huggingface dataset:
Golgi membrane-associated degradation pathway in yeast and mammals (2016)
Figure 6

Figure 6. Induction of GOMED in Atg7-deficient pancreatic β-cells

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. A representative AP-like structure containing insulin granule is shown in (A). An AL-like structure containing insulin granules is shown in (B). Scale bars = 0.2 µm. (C, D) GOMED structures containing Golgi vesicles. An AP-like structure containing multiple Golgi vesicles (arrows) (C) and an AL-like structure fusing with a lysosome (arrowhead) (D) are shown. Scale bar = 0.5 µm. A magnified image of the dashed rectangle is shown in the inset. Scale bar = 0.2 µm. AP-like structure contained double-membrane. (E) The total number of AL-like structure was calculated from the EM images of WT and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min. (F) The total number of GOMED structure was calculated from the EM images of Atg7 KO β-cells after glucose deprivation in the absence or presence of bafilomycin A1 (10 nM). Error bars indicate s.e.m. (n = 109−116). *p < 0.01 vs the value of 60 min. (G−J) EM analysis showed the induction of mitophagy and crinophagy/SINGD. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing mitophagy in Atg7 KO β-cells and crinophagy/SINGD in WT β-cells are shown in (G) and (I), respectively. Arrowhead (G) and white arrow (I) indicates mitophagy and crinophagy/SINGD, respectively. Arrows indicate insulin granules (I). Scale bars = 0.5 µm. (H, J) The number of structures representative of mitophagy (H) and crinophagy/SINGD (J) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min. (K) The number of (pro)insulin granule-containing AL-like structure was calculated from the EM images of wild-type (WT) and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.
Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization (2016)
Figure 1

Figure 1. NLK phosphorylates YAP.

A. NLK but not kinase-negative NLK (Flag-NLK-KM; K155M) caused mobility shift of EGFP-YAP. EGFP-YAP was transfected with empty vector, Flag-NLK-WT, or Flag-NLK-KM into HEK293T cells. Mobility shift was shown by immunoblotting.

B. NLK phosphorylates YAP in vitro. Flag-NLK-WT, or Flag-NLK-KM was immunoprecipitated with anti-Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST-YAP in the presence of [32P-ATP]. Phosphorylation of GST-YAP and autophosphorylation of NLK were examined by autoradiography (top panel), and Coomassie Blue staining showed similar levels of GST-YAP and Flag-NLK (bottom panel).

C. NLK interacts with YAP at the endogenous level. Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti-YAP or anti-NLK antibody.
Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization (2016)
Figure 2

Figure 2. YAP phosphorylation at Ser128 by NLK attenuates phosphorylation at Ser127.

A. Conservation of phosphorylation sites for LATS1/2 and NLK. Amino acid sequences of different species of YAP, Yorkie (in Drosophila), and TAZ (YAP paralog) were aligned. NLK is MAPK (mitogen-activated protein kinase)-type kinase, which prefers proline-directed motif (S/TP).

B. NLK-WT but not NLK-KM reduces Ser127 phosphorylation of endogenous YAP. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. **P < 0.01. Student t-test was used for statistical analysis.

C. Deficiency of NLK leads to increased YAP phosphorylation at Ser127. Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. *P < 0.05 and **P < 0.01. Student t-test was used for statistical analysis.

D. Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure.

E. Substitution of Ser127 with alanine enhances phosphorylation at Ser128, and vice versa. Cell lysates from HEK293T cells transfected with empty vector (-), EGFP-YAP, EGFP-YAP-S127A, or EGFP-YAP-S128A were immunoblotted with antibodies shown in the figure.
Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization (2016)
Figure 3

Figure 3. NLK promotes nuclear localization of YAP by inhibiting interaction between YAP and 14-3-3.

A. Ectopic expression of NLK attenuates interaction between YAP and 14-3-3. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-HA antibody and immunoblotted with anti-GFP antibody (top two panels). Whole cell lysates (WCL) were immunoblotted with antibodies shown in the figure (bottom three panels).

B. Deficiency of NLK leads to increased interaction between YAP and 14-3-3. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-YAP antibody and immunoblotted with anti-14-3-3 antibody (top two panels). WCL were immunoblotted with antibodies indicated in the figure (bottom three panels).

C. Phosphorylation status of Ser127 or Ser128 determines interaction between 14-3-3 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibodies (top two panels). Lysates were immunoblotted with anti-HA or anti-Flag antibodies (bottom two panels). “n.s.” arrow indicates a non-specific band also visible in the non-transfected lane.

D. Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP. Immunofluorescence analysis was performed in HeLa cells transfected with EGFP-YAP, EGFP-YAP-S127A, EGFP-YAP-S128A, or EGFP-YAP-S128D at high cell density. Figure shows a representative of multiple areas. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm.

E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools (bottom panel). Figure shows a representative of multiple areas. Nuclei were stained with DAPI. Scale bars: 20 μm. Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools (top panel).
Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization (2016)
Figure 4

Figure 4. Subcellular localization and protein level of NLK are regulated by cell density.

A. Localization of endogenous YAP and NLK in low (LD) or high density (HD) MCF10A (upper panel) or NIH3T3 cells (bottom panel) was examined by indirect immunofluorescence. Nuclei were stained with DAPI. Scale bars: 20 μm.

B. Level of NLK decreases in a cell density-dependent manner. Lysates from low (LD) and high density (HD) of MCF10A, HEK293T, and NIH3T3 cells were immunoblotted with antibodies indicated in the figure.

C. Level of endogenous NLK mRNA is unaffected, whereas the expression of YAP target genes was reduced, at high cell density. Quantitative real-time PCR analyses for expression of AMOTL2, CTGF, CYR61, and NLK in low (LD) and high density (HD) NIH3T3 cells were performed. Quantification of AMOTL2, CTGF, CYR61, and NLK mRNA was normalized with the level of Gapdh. Data represent average values from a representative of multiple experiments performed in triplicate. Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05 and ***P < 0.005. Student t-test was used for statistical analysis.
Phosphorylation by NLK inhibits YAP-14-3-3-interactions and induces its nuclear localization (2016)
Figure 5

Figure 5. NLK regulates transcriptional activity of YAP.

A. Knockout of NLK reduces the interaction between endogenous YAP and TEAD. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-TEAD1 antibody and immunoblotted with anti-TEAD1 antibody or anti-YAP antibody (top panel). Whole cell lysates (WCL) were immunoblotted with antibodies indicated in the figure.

B. Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies in the figure.

C. Downregulation of nemo with RNAi achieved in the posterior compartment of wing imaginal discs (co-expression of GFP serves to mark the posterior compartment) using the hh-Gal4 driver results in decreased expression of the Yorkie target gene DIAP1. Two different RNAi constructs produce similar effect. Panels below show Z-stacks of magnified areas around the A/P border (left). Quantification of DIAP1 levels achieved upon posterior-downregulation of nemo, measured as normalized posterior-to-anterior levels and compared to similar expression of GFP. Data are present as mean ± sem, n=8 discs. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.

D. Yorkie is epistatic to Nemo: co-overexpression of the constitutively active mutant yki[S168A] together with nmo-RNAi produces typical yki[S168A] phenotypes: severe overgrowth of the affected area with concomitant increase in yki target gene expression (DIAP1). Due to lethality of the hh-Gal4-driven co-overexpression, another posterior driver en-Gal4 was used instead; anti-Hh staining (green) was used to mark the posterior compartment (left panel). Quantification posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 and UAS-yki[S168A]; en-Gal4, presented as in (C); n= 3-6 discs. Posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 are also significantly different from control UAS-GFP; en-Gal4 discs (right panel). Data are presented as mean ± sem. ***P < 0.005. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.

E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids (“EV” stands for “empty vector”), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel). Lysates were immunoblotted with antibodies indicated in the figure (right-bottom panel). Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 200 μm.
Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
Figure 4

Fig. 4: Cyclin A2 is essential to trigger mitotic entry in G2 phase

(A) Time-lapse images of PCNA-mRuby tagged A2dd cells. The imaging sequence was started at the time of Doxycycline addition, and degron activity was triggered three hours later by addition of IAA and Asv or PBS (indicated by dashed line). Time is shown as hh:min, scale bar equals 10µm. For further analysis cells were chosen that had dissipated their PCNA foci before the addition of DIA to ensure that cyclin A2 degradation was triggered in G2 phase.

(B) Single-cell analysis of 40 cells per condition treated as described in (A), each line represents the timing of G2/M/G1 progression for a single cell.

(C) A2dd cells were pulsed with EdU to label S-phase then protein degradation was induced by DIA addition for 5h and before fixation and Edu/CENPF/DAPI staining. Cells that were in G2 at the time of DIA or mock treatment were identified as CENPF positive, EdU negative. Mitotic cells were excluded based on nuclei morphology. The bar plots show mean percentage of EdU negative/CENPF positive cells of three repeats, error bars indicate standard deviation.

(D) Images from time-lapse sequence of mitosis showing cell division in controls and after addition of 0.5µM PD-166285 in DIA treated A2dd labelled with SiR-DNA (time is indicated as hh:min, the scale bars represent 20µm).

(E) Accumulative index of mitotic entry measured by time-lapse microscopy in A2dd cells following mock or DIA treatment and addition of Wee1 inhibitors MK1775 (1µM) or PD-166285 (0.5µM). Asynchronous cells were DIA treated for four hours and then imaged for six hours in five-minute intervals. (Data from 3 repeats, n> 500 cells per condition, standard deviation is indicated by shaded area).

(F) Quantification of metaphase duration in A2dd cells treated with DIA and Wee1 inhibitors as indicated (n>50 per repeat, the boxplot indicates median, first and third quartile and minimum/maximum values.)

(G) A2dd cells were transfected with Ctr or B55α/δ siRNA. 48 hours after transfection the cells were treated with Thymidine for 24 hours and then released. 10 hours later the cells were probed for B55 depletion by immunoblotting using a pan-B55 monoclonal antibody.

(H) Lack of B55 partially rescues mitotic entry in DIA treated A2dd cells. Cells transfected with Ctr siRNA or B55α/δ siRNA as in (B) were released from Thymidine, mock or DIA treated and analysed by widefield live-cell imaging eight hours after the release. The cells were imaged every 10 minutes for 15 hours and manually scored for mitotic index. The lines represent mean values of three independent experiments and standard deviation is indicated by the coloured areas (n>100 per experiment).
Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
Figure 5

Fig. 5: Nuclear cyclin B1 partially compensates for cyclin A2

(A) FACS plots from EdU pulse-chase experiment. Cells were DIA or mock-treated for four hours and then labelled with EdU for one hour. The EdU was then washed out and samples were taken every three hours for 12 hours and analysed by EdU/PI staining and FACS. The top left panel shows the gates that were used for analysis. The quantifications shown in the panels to the right are from three independent repeats of this experiment.

(B) Quantification of cells progressing into Gate 1 (Top panel, new S-phase cells) and Gate 2 (Bottom panel, G2 cells) over time. The lines represent the mean of three experiments, the coloured areas indicate the standard deviation.

(C) Relative changes in the percentage of EdU positive cells in gates 3,4 and 5 (G1/early-S, mid S and late S/G2 phase). The bars indicate the relative mean values from three independent experiments, the error bars represent the standard deviation.

(D) Cyclin A2dd cells with inducible CycB1-YFP (B1-WT) and CycB1-YFP-NLS (B1-NLS) were analysed for DIA induced cyclin B1 expression / cyclin A2 depletion. Samples were collected at indicated time points and probed by immunoblotting with cyclin B1 and cyclin A2 antibodies (* indicates non-specific band).

(E) Quantifications of EdU pulse chase data as in (C) in DIA treated cyclin A2 cells expressing B1-WT, B1-NLS or YFP as indicated. The bars indicate the relative mean values from three independent experiments, the error bars represent the standard deviation.

(F) Accumulative mitotic index measurement of cyclin A2 cells following cyclin A2 depletions and induction of indicated cDNAs. The lines represent the mean of three experiments, the coloured areas indicate the standard deviation.

(G) Quantification of metaphase duration in A2dd cells treated with DIA following induction of indicated proteins (n>50 per repeat, the boxplot indicates median, first and third quartile and minimum/maximum values).

(H) FACS plots from EdU pulse-chase experiment in A2dd cells following induction of indicated genes. Data are representative example for quantifications shown in (E).

(I) Immunoprecipitation of YFP from extract from A2dd following induction of YFP (Y), CycB1-YFP (BY) and CycB1-YFP-NLS (BYN). The samples in the odd lanes (IAA/Asv-) were treated with 1µg/ml doxycycline alone, while samples in even lanes (IAA/Asv+) were treated with Auxin and Asv to induce cyclin A2 depletion. Total cell extract and immune-precipitates were probed by immunoblotting with the indicated antisera.
Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
Figure 7

Fig. 7 Mitotic Greatwall, ENSA and PP1 regulation is independent of cyclin B

(A) Quantification of Greatwall, ENSA/ARPP19 and PP1 phosphorylation based on phospho-proteomic data.

(B) Confirmation of ENSA/ARPP19 S67 phosphorylation in mitotic extracts from Ctr and DIA treated cells, before and after ENSA/ARPP19 siRNA depletion.

(C) Dynamics of mitotic substrate dephosphorylation triggered by Cdk1 inhibition in control and DIA treated B1dd/B2ko cells. The cells were enriched in mitosis by Thymidine release and proTAME/Apcin treatment and treated with 1µM Flavopiridol to trigger mitotic exit. Dephosphorylation was measured by probing the immunoblots with anti Cdk1 substrate (anti-pSP) antibody.

(D) Three repeats of the experiment in (C) were quantified and corrected based on the corresponding GAPDH intensity values. The bar plots show the mean of three experiments and the standard deviation is indicated by error bars.

(E) Dynamics of Greatwall, PP1, and Cdc27 dephosphorylation following Cdk1 inhibition in proTAME/Apcin treated mitotic cell extracts from Ctr or DIA treated cells. Cell extracts were prepared at the indicated times following treatment with 1µM Flavopiridol and analysed by immunoblotting (* indicates non-specific band).
Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B (2020)
Figure 8

Fig. 8 Greatwall and cyclin B synergise in triggering NEBD but not prophase

A) Representative images from live-cell imaging of siRNA transfected B1dd/B2ko cells (FusionRed-H2B in red, SiR-Tubulin in white, scale bar = 10µm). Bar-plot panels on the right show single-cell analysis of 10 cells manually scored for length mitosis pre-NEBD and post-NEBD. Entry into prophase was scored by cell rounding and NEBD was identified by influx of Tubulin in the nucleus.

(B) Widefield Imaging. DIC (Grey), SiR-DNA (b/w), of ENSA/ARPP19 siRNA transfected DIA treated B1dd/B2ko cells. Time is indicated in hh:min, scale bar = 10µm.

(C) Panels of representative immunofluorescence images of siRNA transfected and P/A treated cells treated as indicated, scale bar = 10µm.

(D) Quantification of mitotic index of siRNA transfected P/A synchronised cells (See Fig. 4B for representative images) (mean value of three repeats, n>500 per repeat, error bars indicate standard deviation).

(E) Quantification of mitotic B1dd/B2ko cells following siRNA transfection to deplete Greatwall (siGwl) or ENSA/ARPP19 (siE/A) and/or DIA treatment. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n>50 per repeat, error bars indicate standard deviation).

(F) As in (E) quantification of control and DIA treated mitotic B1dd/B2ko cells following siRNA transfection to deplete Greatwall and B55α/δ. Frequencies of mitotic cells with intact (blue) and disassembled (red) Lamin A/C staining are plotted (mean value of three repeats, n>50 per repeat, error bars indicate standard deviation).

(G) Intensity of pSP-antibody staining in siRNA transfected and DIA treated B1dd/B2ko cells. The swarm-blots classify data from mitotic cells with intact (blue) or disassembled (red) Lamin A/C.

(H) As in (G) Intensity of anti-Lamin A/C pS22 antibody staining in siRNA transfected and DIA treated B1dd/B2ko cells.

(I) Same as in (G) and (H), data for centrosome distance in mitotic cells. Data for G-I are from three repeats, n>50 per repeat, the bars indicate median, first and third quartile as well as minimum and maximum values, data are from three repeats, n=50 cells per repeat).
Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene (2021)
Figure 2

Figure 2. Cas9-targeting of essential NUP58 exons generates hypomorphic alleles.

Determination of maximum growth rate (Vmax) using a label-free cell count-based proliferation assay. Representative growth curves of WT and NUP58 mutant clone F are plotted using a semi-log scale displaying total numbers of cells over time. Growth rates were calculated across the entire duration of the experiment using a sliding window of three timepoints.

Representation of the highest growth rates (Vmax) for each independent cell line. Error bars indicate mean with SD (Welch's t test; B, C, D, F and E ****P ≤ 0.00001, ns = non-significant). Growth rates of mutants clones were compared to the untransfected (WT) cell line seeded in the same plate. Each dot represents a technical replicate (as plotted from left to right: WT n=62, B n=63, C n=61, D n=63, WT n=84, F n=80, E n=82, WT n=90, A n=78) of one independent experimental replicate performed.

Quantification of active nuclear import of asynchronous controls or mutant clones using an NLS-3XmCherry as reporter. Ratio of the cytoplasm vs nucleoplasm fluorescence intensity is plotted. Only cells with comparable overall fluorescence intensity are reported (see extended M&M for details). Dots represent analysed cells (WT n=39, B n=22, C n=50, D n=34, E n=34, F n=9, A n=42) of one independent experimental replicate performed. Error bars indicate mean with SD (Welch's t test; ns = non-significant, B, C, D, E ****P≤ 0.00001, F ***P = 0.0001).

Representative images of analysed WT (top) and clone D (bottom) NLS-3xmCherry stable cells. Mean intensity of fluorescence for cytoplasm and nucleoplasm for each cell are labelled. Scale bar, 20 μm.

Western blot for NUP58 protein expression in mutant clones (B-E) and control cells (clone A and WT). Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Please see Figure S2A for detailed description of the used antibodies. Actin was used as loading control.
Non-genetic and genetic rewiring underlie adaptation to hypomorphic alleles of an essential gene (2021)
Figure 4

Figure 4. Alternative exon usage resulted in novel NUP58 protein variants.

Sashimi plots indicating combined exon usage of NUP58 mRNA in HAP1 WT and mutant clones across four biological replicates. Numbers of reads per exon-exon junction are indicated below introns. WT canonical isoform is colored in black; mutant isoforms in grey. NUP58 gene structure is reported over the sashimi plot; red asterisks and exons highlight the positions of the identified mutations.

Western blot for NUP58 protein expression in mutant clones and control cells. Whole-cell lysate hybridized with customized antibody 1 (Ab1, top panel) and antibody 2 (Ab2, bottom panel). Blots were loaded with wild-type HAP1 cell line (WT), empty vector (EV), non-targeting siRNA (siNT) and NUP58 targeting siRNA (siNUP58), followed by NUP58 mutant clones (C1 / C2 / C3 / C4 in left panels, C5 / C6 in right panels). Actin was used as loading control. Asterisks indicate possible NUP58 alternative isoforms.