EMBO/soda-nel huggingface dataset:
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 2

Figure 2. LATS1/2 double knockout drives YAP nuclear localization, crypt hyperplasia, and a long/flat Wnt gradient.

A) Murine small (top) and large (bottom) intestines isolated from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Immunostaining for YAP and Ki67 shows a gradient of YAP expression along the crypt-villus axis in controls, with nuclear YAP and Ki67 positive cells restricted to the crypt base (representative images from n=5 mice). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show an enlarged crypt compartment after 7 days with strongly nuclear YAP immunostaining in all epithelial cells and an expanded proliferative zone marked by Ki67 positive cells. Note the gradient of YAP expression levels is maintained along the crypt-villus axis (representative images from n=5 mice for each genotype). (Right) Immunostaining for the Paneth cell marker Lyz reveals loss of this marker from the crypt base. (Bottom right) Q-PCR analysis of Wnt pathway target genes reveals that Lats1/2 dKO causes a mild reduction in Lgr5 expression, with complete loss of Olfm4.

B) Axin2 mRNA expression was measured by RNAscope (red) and found to be increased in both intensity and uniformity of staining along the crypt-villus axis in Lats1/2 dKO after 3 days post i.p. injection (dpi) with tamoxifen to induce the homozygous deletion of Lats1 and Lats2. At 7dpi, Axin2 mRNA levels remain uniform along the crypt-villus axis in Lats1/2 dKO animals, although their total level has declined compared to 3dpi.

C) Schematic diagram showing alteration of the Wnt signalling gradient upon Lats1/2 dKO in the intestine, with crypt expansion being accompanied by an elongation/flattening of the Wnt signalling gradient, whose elevated level gradually declines over time.

D) Q-PCR analysis of YAP-TEAD and their target genes Ctgf and Cyr61 reveals that Lats1/2 dKO causes a strong induction of Ctgf and Cyr61 expression, as expected, as well as a strong induction of the common Wnt and YAP target gene CyclinD1.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 3

Figure 3. TEAD1/4 mRNA expression is progressively expanded in Lats1/2 dKO intestines.

A) Q-PCR analysis of TEAD1-4 mRNA expression in control versus Lats1/2 dKO intestines at 7-days post i.p. injection with tamoxifen. Note the strong increase in TEAD1 and TEAD4 mRNA levels in particular, which indicates that these are both target genes of YAP signalling.

B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)

C) Q-PCR analysis reveals a progressive increase in the YAP-TEAD target gene Ctgf in Lats1/2 dKO intestines at 3-days and 7-days post i.p. injection with tamoxifen, as well as similarly increased TEAD1 and TEAD4 expression, confirming that TEAD1 and TEAD4 are YAP-responsive genes. Notably, Axin2 and TEAD2 exhibit a mild but progressive decline over 3 to 7 days after Lats1/2 deletion, consistent with Axin2 being a known Wnt-specific target gene, and with Wnt signalling remaining active in Lats1/2 dKO intestines.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 4

Figure 4. Wnt signalling induces TEAD1/2/4 mRNA expression in the intestine.

A) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (wild-type) and ApcMin mutant tumours from the small intestine (top) and colon (bottom). All three genes were strongly induced within the ApcMin mutant tumours, with TEAD2 being most strongly induced in colon ApcMin tumours (n=3 animals with multiple tumours within the small and large intestines).

B) Q-PCR analysis of control and Apc5 mutant murine intestinal organoids in vitro, control and ApcMin mutant tumour samples in vivo, and control and Apcfl/fl knockout hyperplastic crypts in vivo. In all cases, TEAD1/2/4 mRNA expression was strongly elevated upon loss of Apc function, while TEAD3 mRNA expression was unaffected. (RNA was isolated from 6 individual tumours).

C) Antibody staining for TEAD4 reveals a gradient of expression along the crypt-villus axis in both the small and large intestine, with uniformly high expression in colorectal adenomas, similar to SOX9.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 5

Figure 5. YAP integrates Wnt signalling and mechanical stimuli in intestinal organoids.

A) YAP and Ezrin immunostaining in control murine intestinal organoids cultured for 1-5 days as indicated. Note the initially cytoplasmic localisation of YAP, which becomes nuclear localised after 3 days.

B) YAP immunostaining of 3-day old wild type organoids treated with DMSO (Control) or Porcupine inhibitor LGK974 at 5µM. Wnt signalling inhibition upon Porcupine inhibitor treatment decreases YAP levels without affecting YAP subcellular localisation. Quantification of YAP protein levels is shown below, with statistical significance in an unpaired 2-tailed t-test.

C) Wnt activation in Apc5 homozygous mutant organoids drives a drastic shift in organoid morphology from budding structures to cystic spheres and increases the YAP protein level. Scale bar is 250µm. n > 10 organoids in each experiment. Quantification of YAP protein levels from crypt and villus regions of control organoids as well as from the regions bounded by rectangles in Apc5 organoids are shown on the right, with statistical significance in an unpaired 2-tailed t-test.

D) Wild-type organoids, at early stages of development (1-2d) prior to crypt budding, form small spheroids that also exhibit either a columnar or a stretched epithelium where YAP is cytoplasmic or nuclear, respectively.

E) Apc5 organoids (5d) exhibit either a columnar or a stretched epithelium where YAP is cytoplasmic or nuclear, depending on the shape of the cells.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 6

Figure 6. In both wild-type and Apc5 organoids, YAP nuclear localisation is prevented by a Src family kinase inhibitor

A) Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4h of treatment in organoids cultured for 3d. n > 10 organoids in each experiment.

B) Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** p <0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired student's t-test.

C) Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. Q-PCR analysis of the YAP-TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired student's t-test.

D) Src family kinase inhibition via Dasatinib or eCF506 treatment in VillinCreERt Lats1/2 dKO organoids (induced in vitro with 4-OHT) fails to cause relocalisation of YAP to the cytoplasm after 4h treatment in culture. n > 10 organoids in each experiment.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 7

Figure 7. Irradiation-induced nuclear localisation of YAP involves Src family kinase signalling.

A) Mouse intestinal organoids immunostained for YAP (green). Note the mostly cytoplasmic localisation of YAP at 48h in the organoid. Irradiation with 4Gy of X-rays drives strong nuclear localisation of YAP in many cells, which is reversible by treatment with the Src family kinase inhibitor Dasatinib. The Dasatinib treated organoids exhibit increased cell death after irradiation, as indicated by pyknotic nuclei (white arrow). n > 10 organoids in each experiment. This experiment employs the same control batch of 2-3d organoids also shown in Figure 6A.

B) Control VillinCreERt mouse small intestine immunostained for YAP shows a gradient of expression with mostly cytoplasmic localisation. Irradiation with 14Gy of X-rays drives strong nuclear localisation of YAP along the entire crypt-villus axis. Deletion of Src within VillinCreERt Srcflox/flox intestines prevents YAP nuclear localisation and normal regeneration after 14Gy irradiation. Arrows point to villar cells with nuclear YAP localisation. Lower panels show high-mag views of a different example than those shown in upper panels.
Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration (2021)
Figure 8

Figure 8. Irradiation induces YAP-TEAD activity to drive crypt expansion, which also requires Wnt signalling.

A) Control and irradiated murine small intestines analysed with RNAscope in situ hybridisation for Myc mRNA (black arrows point to mRNA granules in the control) and by immunostaining for Sox9 protein reveals an expansion in the expression domain of both genes. Nuclear localisation of beta-catenin is readily detectable after irradiation. n = 3 samples per condition, quantification shown in the insets, error bars = 1 SD. Statistical analysis was performed with an unpaired student's t-test. Similar results were obtained at 3dpi and 5dpi.

B) Q-PCR analysis of YAP, TEADs and their target genes Ctgf and Cyr61 over a 4 days post irradiation time course at 12Gy. Note the strong upregulation of TEAD4, Ctgf, and Cyr61. In contrast, Axin2, TEAD2 and Sox9 mRNA expression levels are only moderately altered, confirming that Wnt signalling remains active upon irradiation-induced YAP-TEAD activation. n = 4 biological replicates per time point. Statistical analysis was performed with unpaired t-test compared to the control 0Gy condition, * p<0.05, ** p<0.01, ***p<0.001.

C) Control and irradiated murine small intestines analysed by immunostaining for Sox9 protein reveals that treatment with Porcupine inhibitor LGK974 strongly reduces the Sox9 expression domain induced by 3 days after 12Gy irradiation. Phosphorylated Src immunostaining reveals an upregulation by 3 days after irradiation which is also lost upon treatment with Porcupine inhibitor LGK974 (n= 4 animals in each condition).

D) Q-PCR analysis of Wnt and YAP target gene expression in intestinal crypts isolated at 1 day after 12Gy irradiation. Note the inhibition of Axin2, Sox9, YAP, Ctgf, Cyr61, and TEAD family expression upon treatment with Porcupine inhibitor LGK974. The mild reduction of Sox9 mRNA expression by LGK974 at 1 day after irradiation (1dpi) becomes more pronounced at 3 days after irradiation (3dpi). n = 4 biological replicates per condition, statistical analysis was performed with Student's 2-tailed t-test, error bars = 1 SD, * p<0.05, ** p<0.01, ***p<0.001.
Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores (2022)
Figure 3

Figure 3. A farnesyl-binding pocket in the ROD propeller

(A) Schematic representation of HsSpindly. The position of a CC1 box (red) and of the Spindly box (green) required for binding Dynein:Dynactin are shown, together with the position of relevant predicted coiled-coil regions.

(B) Cartoon representation of the 7-bladed ROD β-propeller (indicated as blades 1-7) modelled with AF2. The positions of blade 2, blade 3, and an α-helical hairpin representing an insertion between strands β3c and β3d are indicated. The four strands of each blade are indicated as A-D, with A and D being the innermost and outermost strands. The circle represents the entry point of the farnesyl binding pocket.

(C) Surface representation of the ROD β-propeller. The circle is in the same position shown in panel A. The position of Leu120 is shown in yellow. This residue was targeted by photoactivatable crosslinker groups introduced in the farnesyl group attached to Cys602 of Spindly (Mosalaganti et al., 2017).

(D) The position of a modelled peptide corresponding to the C-terminal region of Spindly as predicted by AlphaFold2 (Jumper et al., 2021; Tunyasuvunakool et al., 2021) with a farnesyl moiety modelled on Cys602.

(E-F) A farnesyl moiety, shown in cyan spheres together with a few C-terminal residues of a modelled Spindly peptide, fitted snugly into a pocket lined exclusively by the side chains of several hydrophobic residues.

(G) Size-exclusion chromatography profiles and corresponding SDS-PAGE of the indicated samples. Two mutant RZZ complexes containing mutations in ROD L110F-L119F-L120K or I191M are indicated respectively as FFK and I/M. Note that in the bottom SDS-PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker. For all other shown SDS-PAGE gels, the marker precedes the first fraction.
Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores (2022)
Figure 4

Figure 4. MPS1 and Spindly promote corona assembly

(A) Confocal fluorescence microscopy-based filamentation assay at 561 nm shows mChRZZSF (4 µM), but not mChRZZ (4 µM) or mChSF (4 µM) as controls, forms filaments at 30ºC. Scale bar: 5 µm. Two technical replicates were performed.

(B) Coomassie and ProQ Diamond-stained gels on mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM. These were precisely the samples studied in experiments in panel C.

(C) A filamentation assay as in A demonstrates sufficiency of MPS1 phosphorylation for filamentation. Experiments for panels B and C were technically replicated three times.

(D) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM reversine, 3.3 µM nocodazole, and 10 µM MG132 for 1 hour and imaged while in mitosis. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed.

(E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm.

(F) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.

(G) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole for an additional hour. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed.

(H) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G. Red lines indicate median with interquartile range.

(I) Levels of CENP-E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly. Three biological replicates were performed.

(J) Scatter dot plots representing normalized total area of the CENP-E and Zwilch signals, normalized to the RNAi negative control, in the indicated number of cells from the experiment shown in panel I. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.
Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores (2022)
Figure 5

Figure 5 Role of the ROD N-terminal region in filamentation

(A) A predicted N-terminal extension (green) of the ROD β-propeller (red), slightly rotated from the view in B. The propeller proper begins with strand β7d and ends with strand β7c, which leads into the helical domain. The N-terminal extension augments the sixth and seventh blades with external β-strands (β6e and β7e). The position of T13 and S15 on the extension is shown.

(B) The AF2 model confidence score (pLDDT, displayed blue to red through green from highly to poorly reliable) highlights regions of the model predicted with high or poor confidence, respectively (Jumper et al., 2021; Tunyasuvunakool et al., 2021).

(C) MPS1-induced filamentation experiments demonstrate that the phosphomimetic T13E/S15E ROD mutant bypasses the filamentation blockade induced by the MPS1 inhibitor reversine. The T13A/S15A mutant prevents MPS1-induced filamentation altogether.

(D) N-terminal deletion mutant of ROD (∆15 and ∆26) removing the first 15 or 26 N-terminal residues that include the MPS1 phosphorylation sites Thr13 and Ser15 are unable to form filament in presence of MPS1 at 20ºC, but can form filaments upon mildly heating to 30ºC.

(E) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS-PAGE separation of reactions.

(F) Filamentation assays with the indicated combinations of 8 µM mChRZZSF, MPS1 (1 µM), and lambda phosphatase (0.4 mg/ml) in presence of 10 µM reversine. Dephosphorylation reactions were carried out on already formed filaments (see Methods). Samples were imaged by confocal microscopy. Dephosphorylation does not dissolve already formed filaments.

(G) RZZ and RZZS complexes were reconstituted with the ZwilchE422A/D426A mutant and tested for filamentation at 20ºC in presence of MPS1 or upon mildly heating to 30ºC. These experiments are also displayed in Figure EV4A.

(H) Surface representation of the RZZ model depicting the position of ZwilchE422 and ZwilchD422 (in purple) and the positions of the ROD N-terminal region (green) and the highly conserved ZW10 N-terminus.

Data information: Scale bars in panels C, D, F, G = 5 µm. Two technical replicates for panels C, E, F, and G were performed. Three technical replicates for panel D were performed.
Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores (2022)
Figure 7

Figure 7 Influence of Spindly on corona assembly and kinetochore recruitment

(A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction. Two technical replicates were performed.

(B) MPS1-induced filamentation experiments on the indicated mChRZZSF complexes (4 µM, further diluted to 0.5 µM for imaging) using a confocal spinning disk fluorescence microscope at 561 nm. Scale bar: 5 µm. Two technical replicates were performed.

(C) Schematic of the cell synchronization and imaging experiment shown in D. After electroporation, cells were allowed to recover for 8 hours. Subsequently, cells were synchronized in G2 phase with 9 µM RO3306 for 15 hours and then released into mitosis by inhibitor washout. Before fixation, cells were treated with 3.3 µM nocodazole for 1 hour.

(D) Representative images of fixed HeLa cells electroporated with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly by RNAi. Spindly localization was detected with an antibody against the C-terminal region of Spindly. Corona expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI to stain for DNA. Scale bar: 10 µm. Experiments were replicated three times.

(E) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.

(F) Cells treated like in C-D were electroporated with the indicated mChSpindly constructs. Corona expansion was evaluated through the appearance of Zwilch. Experiment replicated three times. Scale bar: 10 µm.

(G) Kinetochore intensities were quantified like in panel E. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.

Figure EV Legends
A genome-wide RNAi screen reveals essential therapeutic targets of breast cancer stem cells (2019)
Figure 1

Figure 1. A genome-wide RNAi screen for the identification of bCSC-fate regulator. A. Schematic representation of RNAi screening strategy. B. Detection of ALDHbr cells by high content screening. The algorithm first detect nuclear region of interest (ROI) in the Hoechst channel (rainbow areas). Then, it computes the averaged ALDH signal in the ROI to identify ALDHbr cells (red nuclear ROIs) and calculate their proportion. C-D. Example of candidate genes whose silencing modified the ALDHbr cell proportion without inducing a massive cell death (cell viability>20%, B-score>│2.58│). Data represent mean ±SD (n=3). E. Representative images of the high-content screening captures. ALDEFLUOR cellular staining is represented in green. ALDHbr cells are labelled in red. F. Volcano plot of the hits. Scatter points represent genes, the x-axis is the log2 fold change of the proportion of ALDHbr cells compared to the control, and the y-axis is the B-score. Genes targeted by a siRNA inducing a significant reduction (green dots; B-score>2.58, log2 FC<1) or increase (red dots; B-score>2.58, log2 FC<1) of the ALDHbr cell proportion are highlighted.