EMBO/soda-nel huggingface dataset:
Identification of Cdk targets that control cytokinesis (2014)
Figure 1

Cytoplasmic Cdc14 is dispensable for mitotic exit

cdc14‐3 temperature‐sensitive cells reconstituted with CDC14 or CDC14‐NLS were released from a G1 arrest at the restrictive temperature and stained for Cdc14 and the spindle (Tub1) once cells had reached late anaphase.
Wild‐type, cdc14‐3, +CDC14 and +CDC14‐NLS cells were released from an α‐factor‐induced G1 arrest and re‐arrested in the next G1. Samples were harvested at the indicated time points and stained with DAPI and for the nucleolar protein Nop1 (n > 100). Photographs of examples of cells without (top) and with (bottom) rDNA segregation defects at 80 min after release are shown together with quantification over time.
Cells from the experiment in (B) were stained for the spindle (n > 100). Cells without (top) and with (bottom) anaphase spindles at 100 min after release are shown together with their quantification over time.
FACS analysis of DNA content and Western blot analysis of cell cycle markers of the samples from (B). Tub1 served as a loading control.

Data information: Scale bars, 5 μm.
Identification of Cdk targets that control cytokinesis (2014)
Figure 2

Cytoplasmic Cdc14 is required for cytokinesis

+CDC14 and +CDC14‐NLS cells were grown at 35.5°C for 5 h and sonicated, and differential interference contrast (DIC) images were acquired.
Cells from a synchronous culture (as in Fig B) were treated with zymolyase to remove the cell wall. The fraction of remaining budded cells is shown over time (n = 100).
Cells expressing the plasma membrane marker GFP‐Spo2051–91, exemplifying the indicated bud neck phenotypes and stages of cytokinesis.
Bud neck phenotypes of synchronized GFP‐Spo2051–91‐expressing +CDC14 or +CDC14‐NLS cells during the progression of a cell cycle (n > 75).
+CDC14 or +CDC14‐NLS cells expressing Spc42‐YFP and GFP fusions with the indicated cytokinetic protein were synchronized in G1 using α‐factor. Upon release at 35.5°C, cells were imaged every 2 min and the time from anaphase onset to the indicated events was recorded. Error bars represent SD; Student's t‐test was applied to analyze differences between the indicated conditions; **P ≤ 0.01; ****P ≤ 0.0001. For representative image sequences, see Supplementary Fig S2C.

Data information: Scale bars, 10 μm.
Identification of Cdk targets that control cytokinesis (2014)
Figure 3

Cdk downregulation and Cdc14 drive MEN‐independent cytokinesis

MET3pr‐CDC20 cdc15‐as1 cells were arrested in metaphase by Cdc20 depletion and subsequently treated with 1NM‐PP1 to inhibit Cdc15‐as1. Cells were released from the metaphase arrest by Cdc20 re‐induction (left graph), or Cdc14 and/or Sic1 expression were induced from the GAL1 promoter while cells remained arrested (right graph). The fraction of large‐budded cells after spheroplastation (n = 100) was quantified. Representative photographs 120 min after galactose addition are shown. Scale bar, 10 μm.
As (A), the plasma membrane marker GFP‐Spo2051–91 was visualized following Cdc14 induction (n = 100).
Samples from the experiment in (B) were processed for Western blot analysis of the indicated cell cycle proteins. Arrowheads indicate endogenous and exogenous forms of Cdc14. Tub1 served as a loading control. A schematic of downstream processing for the phosphoproteome analysis is shown on the right.
Identification of Cdk targets that control cytokinesis (2014)
Figure 4

Phosphoproteome analysis of Cdc14‐induced mitotic exit

Phospho‐peptide abundance during Cdc14‐induced mitotic exit. Examples of phospho‐peptides that disappear with early (red), intermediate (orange) and late (green) timing or that remain stable (black) are shown. This color coding is used throughout all panels of this figure.
Numbers of unique peptides and corresponding proteins that do or do not contain a minimal Cdk consensus motif are shown, categorized according to timing of disappearance as described in (A).
Protein stability data for proteins corresponding to disappearing phospho‐peptides (blue) and for all other proteins (black).
The fractions of peptides that contain a minimal Cdk consensus motif ([S/T]‐P) within the timing categories shown in (B) were calculated. The ratio and P‐value of enrichment of each category relative to the combined stable/rest category (gray) are shown.
Similar to (D), the enrichment for reported physical Cdc14 interactors is assessed within [S/T]‐P‐containing phospho‐peptides.
Peptides that contain the indicated Cdk consensus sequence motifs and that have disappeared over time are represented as a fraction of all phospho‐peptides containing that motif.
Unique phospho‐peptides of Spa2, as an example, are categorized based on their dephosphorylation timing (graph). Enrichment of clustered timing for all proteins covered by phospho‐peptides was compared to random for Cdk or non‐Cdk consensus site‐containing peptides (table). Fisher's exact tests were used to determine significance.
Enrichment of the indicated gene ontologies among Cdk consensus site‐containing peptides as in (D).
Identification of Cdk targets that control cytokinesis (2014)
Figure 5

Identification of phospho‐regulated cytokinetic factors

Targeting strategy used for creating conditionally phosphorylated proteins. Upon integration of the targeting cassette and expression of the gene of interest (GOI), the targeted protein is C‐terminally tagged with an HA epitope. After Cre recombinase‐mediated excision, Clb2m‐HA is fused to the targeted protein.
Western blot analysis of cells expressing conditional Clb2m (+) or Clb2mΔCdk (Δ) fusions to the indicated proteins, before (−) and 5 h after (+) β‐estradiol treatment. Tub1 served as a loading control. c, control cells.
FACS analysis of DNA content after conditional Clb2m or Clb2mΔCdk fusion to the indicated proteins. Quantification of the fraction of cells with > 2C DNA content is shown in the bar graph for two independent biological replicates, with red dots representing individual and bars representing average values. Bnr1 fusions were included as controls for cytokinetic defects. Student's t‐test was applied to compare matched Clb2m versus Clb2mΔCdk fusion strains. ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Serial dilutions of cells from (C) were plated 6 h following β‐estradiol treatment and grown for 1 day at 36°C.
The length of cell chains containing a common cytoplasm was scored 6 h after β‐estradiol treatment using the GFP‐Spo2051–91 plasma membrane marker (n > 150). c, control cells; scale bars, 10 μm.
Identification of Cdk targets that control cytokinesis (2014)
Figure 7

Inn1 phosphorylation regulates bud neck localization and Chs2 activation

Cyk3‐GFP signal intensity at the bud neck relative to the onset of actomyosin ring contraction (t = 0). The mean and SEM from at least 20 cells are shown.
Inn1‐Clb2m, Inn1‐Clb2mΔCdk and control cells before Clb2m fusion were arrested in G1 using α‐factor, released and harvested 90–100 min later at the time of cytokinesis. Indirect immunofluorescence was used to determine Inn1 bud neck staining and the spindle status. Representative images of full spindles and partially and fully disassembled spindles are shown. nr, non‐recombined cells. Scale bars, 5 μm.
The time from anaphase onset until the bud neck appearance of Inn1 was determined by filming synchronized Inn1‐Clb2m‐GFP and Inn1‐Clb2mΔCdk‐GFP cells containing Spc42‐YFP‐labeled spindle pole bodies. Error bars represent SD.
As in (C), now the persistence of a full actomyosin ring was measured in cells expressing Inn1‐Clb2m, Inn1‐Clb2mΔCdk or their respective non‐phosphorylatable mutants together with Myo1‐GFP and Spc42‐YFP. Error bars represent SD.
Montage of medial view projections of actomyosin rings in Inn1‐Clb2m and Inn1‐Clb2mΔCdk cells. Frames are shown in 2‐min intervals.
Actomyosin ring contraction phenotypes from at least 21 cells filmed as in (E) were blindly scored as having a normal (“constriction”) or aberrant (“disintegration”) cytokinetic phenotype.
Cytokinetic defect and cell viability of Inn1‐Clb2m or Inn1‐Clb2mΔCdk cells in the absence or presence of Chs2V377I. In the bar graph, red dots represent values of duplicate biological replicates with bars representing the average. Significance is calculated using Student's t‐test. 10‐fold spot dilution assays are represented, together with the Western blotting analysis for Inn1 before and after its fusion to Clb2m. Tub1 served as a loading control.

Data information: ‐P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes (2022)
Figure 4

Figure 4. Behavior of Meiosis-regulating Proteins in Spermatocyte-injected Oocytes

A SGO2 localizes at the centromeres of spermatocyte-derived and maternal chromosomes. Oocytes were fixed at metaphase I and stained for SGO2 (green), ACA (centromeres, magenta), and Hoechst33342 (DNA, blue). Images were reconstructed in 3D and viewed from the top and side of the metaphase plate. Note that signals are interpolated in z.

B PLK1 localizes at the kinetochores of spermatocyte-derived and maternal chromosomes. PLK1 (green) localization was investigated as in (A).

C MAD2 kinetochore localization is defective on spermatocyte-derived chromosomes. The localization of the closed (active) form of MAD2 (C-MAD2, green) was investigated as in (A). Note that the kinetochore enrichment of MAD2 was less on the chromosomes in half of the metaphase plate, which likely corresponded to spermatocyte-derived chromosomes, in control and halved oocytes.

D Effect of forced SAC activation. Oocytes expressing MAD1-mNeonGreen-CENP-C (green), together with major satellite-mClover (green) and H2B-mCherry (red), were imaged. Time after the start of imaging (h:mm) is shown. Oocytes were categorized based on anaphase figures (n = 24, 23 oocytes).
Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region (2016)
Figure 2

Figure 2. UbL domains bind with proteasome.

(A) Sketch of UbLRad23-GFP-95 consisting of the UbL domain of S. cerevisiae Rad23, followed by superfolder GFP and a 95-amino-acid-long tail derived from S. cerevisiae cytochrome b2. (B) In vitro degradation of UbLRad23-GFP-95 by purified S. cerevisiae proteasome. The graph plots the amount of substrate over time, estimated by fluorescence intensity monitored by plate-reader (green) or electronic autoradiography of SDS-PAGE gel bands (red), normalized to the initial substrate amount as described in Methods. (C) Michaelis-Menten analysis of UbLRad23-GFP-95 degradation by different concentrations of purified S. cerevisiae proteasome (green, 10 nM; red, 40 nM). (D) UbLRad23 inhibits UbLRad23-GFP-95 degradation. The initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition. (E) The UbLDsk2 domain is a competitive inhibitor of UbLRad23-GFP-95 degradation. Lineweaver–Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain. (C-E) Proteasomal degradation of UbLRad23-GFP-95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods.
Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region (2016)
Figure 3

Figure 3. UbL domains target substrate to proteasome degradation in vitro.

(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.
Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region (2016)
Figure 4

Figure 4. UbL domains target His3 substrates to proteasome degradation in yeast.

(A) Schematic representation of yeast growth assay. The model proteins consisted of an UbL domain and S. cerevisiae imidazoleglycerol-phosphate dehydratase (His3), followed by stop codon (no tail) or a 51 amino acid tail derived from subunit 9 of the Fo component of the Neurospora crassa ATPase (Su9) at the C-terminus. In his3 mutant cells, the absence of His3 protein causes a growth defect when grown on selective media. Stable His3 proteins escape proteasome degradation and rescue the his3 mutant cells from the growth defect. (B) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail, and affected yeast growth under selective condition (+3-AT, -his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 days for imaging.
Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region (2016)
Figure 5

Figure 5. UbL domains target fluorescence substrates to proteasome degradation in yeast.

(A) Fluorescence-based degradation assay in S. cerevisiae. The substrate proteins consisted of a UbL domain, followed by a yellow fluorescent protein (YFP) domain and a disordered tail (51 amino acids derived from subunit 9 of the Fo component of the Neurospora crassa ATPase, Su9). Control substrates lacked the disordered tail. Substrates and a red fluorescent protein were expressed from consecutive constitutive promoters on the same CEN plasmid (YCplac33). (B) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry. UbLRad23 targeted YFP-Su9 protein for degradation. Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells. (C) UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.
Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region (2016)
Figure 6

Figure 6. Degradation of Mdy2 proteins in vitro and in vivo.

(A) Schematic representation of S. cerevisiae Mdy2. Structural models of the N-terminal domain (NTD) (amino acids 1-59 of Mdy2, green, with Get4 in red, PDB ID: 2WPV), the central UbL domain (amino acids 74-148 of Mdy2, grey, PDB ID: 4GOC), the Cterminal dimerization domain (DD, amino acids 152-212 of Mdy2, blue, PDB ID: 2LNZ) of Mdy2. (B) In vitro degradation of Mdy2 proteins by purified S. cerevisiae proteasome. Mdy2 (red solid circles), Mdy2ΔN-95 (blue solid circles) and Mdy2-95 (green solid circles) were degraded. Removing the N-terminal domain of Mdy2 (Mdy2ΔN, black solid circles) or adding its binding partner Get4 (red open squares, dash line) or the proteasome-binding competitor UbLMdy2 (red open diamonds, dotted line) stabilized Mdy2 proteins. When Get4 binding buried the N-terminal domain, addition of a 95 amino-acid tail derived from S. cerevisiae cytochrome b2 at the Cterminus restored degradation of Mdy2 (green open squares, dotted line). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m. (C) Degradation of Mdy2 in vivo. The steady-state accumulation of overexpressed C-terminally HA-tagged full-length Mdy2 and a mutant in which the N-terminal domain was deleted (Mdy2ΔN) in S. cerevisiae, in the absence of Get4, the presence of endogenously expressed Get4 or overexpressed Get4. Overexpressed Get4 was detected through an N-terminal Flag-tag. (D) Steady-state accumulation of endogenous Mdy2 in S. cerevisiae, with deleted (get4∆) or endogenous (wt) Get4 in the presence or absence of the proteasome inhibitor bortezomib. Protein levels were determined by western blotting for the HA-tag, Flag-tag, Mdy2 or Scs2 of SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.
Epithelial response to IFN-γ promotes SARS-CoV-2 infection (2021)
Figure 1

Fig.1 IFN-γ is a strong driver of ACE2 expression in human colon organoids

A) (left) t-sne plot from scRNAseq data of human organoids and cluster assignment of colonic cell populations. Red color corresponds to undifferentiated cells, light green corresponds to surface colonocytes (enterocytes). t-sne plot for selected genes from single cell RNAseq of human colon epithelial cells revealing expression of ACE2 specifically in differentiated surface colonocytes expressing KRT20 and AQP8, which also express the IFN-γ receptor IFNGR2.

B) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of human colon tissue section indicating ACE2 expression in surface colonocytes. Scale bar: 50 µm.

C) Human colon organoids cultured in full medium (FM) condition (upper panel) or FM treated with IFN-γ (lower panel). Left: bright field images of 7-day cultured human colon organoids, scale bar: 100 µm. Right: Immunofluorescence for KRT20 (green; note increased expression upon IFN-γ treatment). Scale bar: 25 µm.

D) Human colon organoids cultured in basic medium (upper panel) or additionally treated with IFN-γ (lower panel). Left: bright field images of organoids that were cultured for 4 days in basic medium and in addition for 3 days with IFN-γ (lower panel) scale bar: 100 µm. Middle: Immunofluorescence labelling for KRT20 (green) Right: Immunofluorescence labelling for E-cadherin (green) to visualize epithelial cell boundaries; insets with higher magnification indicate elongation of epithelial cells that are grown in basic medium and treated with IFN-γ. Scale bar: 25 µm.

(E-I) Comparison of E) KRT20, F) MUC2, G) CHGA, H) LGR5 and I) ACE2 mRNA expression in organoids grown in FM or in basic medium and either untreated or treated with IFN-γ (n=3). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test (more details see Appendix Table S2).

J) Immunofluorescence staining for ACE2 (red) and KRT20 (green) of organoids grown in full medium (upper) or basic medium (lower) and additionally treated with IFN-γ (right), indicating expression of ACE2 in differentiated enterocytes. White arrows point to ACE2. Scale bar: 10 µm
Epithelial response to IFN-γ promotes SARS-CoV-2 infection (2021)
Figure 2

Fig.2 IFN-γ increases infectivity of SARS-CoV-2 in colonic organoids

A) Immunofluorescence for N-protein (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Yellow arrows point to N-protein positive cells. Scale bar: 25 µm.

B) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. basic medium organoids 48 h after infection and normalized to GAPDH (n=6).

C) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium vs. full medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=9).

D) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in basic medium vs. basic medium + IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4).

E) qPCR data displaying the relative virus load of SARS-CoV-2 measured by viral genome quantity in full medium + IFN-γ vs. basic medium+ IFN-γ treated organoids 48 h after infection and normalized to GAPDH (n=4).

(B-E) Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by Student's t-test (more details see Appendix Table S2)

F) qPCR data comparing the relative virus load of SARS-CoV-2 measured by viral genome quantity normalized to GAPDH at 24 and 48 h after infection in full medium (left) and basic medium (right) untreated or treated with IFN-γ, indicating increase in virus load in differentiated and IFN-γ treated conditions (n=8). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test (more details see Appendix Table S2).

G) Electron microscopy imaging of organoids grown in basic medium, pretreated with IFN-γ for 3 days and infected for 48 h with SARS-CoV-2. Upper: Overview showing large virus-loaded vesicles in an epithelial cell. Scale bar: 500nm. Lower: Disintegrated organoid cell containing high virus load, scale bar: 2.5µm, higher magnification to visualize virus particles, scale bar: 100nm.

H) Immunofluorescence for cleaved-caspase3 (red) and E-cadherin (green) 48 h after SARS-CoV-2 infection of human colon organoids cultured in full medium (upper), full medium + IFN-γ (upper right), basic medium (lower left) and basic medium + IFN-γ (lower right). Scale bar: 50 µm. (below) Quantification of apoptotic cells, indicating increased cell death in the differentiated and IFN-γ treated condition after SARS-CoV-2 infection (n=7). Data are presented as mean +/- SD ,*: p≤0.05, **: p≤0.001, ***: p≤0.0001, as determined by one-way ANOVA, followed by Tukey's multiple comparisons test (more details see Appendix Table S2).