EMBO/soda-nel huggingface dataset:
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 5

Figure 5. Wip1 interacts with Smad4 in vivo.

(A, B) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands.

(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm.

(D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test.

(E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein.

(F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results.

(G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 6

Figure 6. Wip1 is a Smad4 phosphatase.

(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.

(B) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1-depleted HEK293T cells, showing that knockdown of Wip1 up-regulates Smad4 phosphorylation at the MAPK site. A bracket denotes phospho-Smad4.

(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.

(D) FGF-induced destabilization of Smad4 is enhanced further in Wip1-depleted animal cap cells as analyzed by western blotting. The amount of reagent injected: Myc-Smad4 (400 pg), FGF8 (100 pg), Wnt8 (400 pg), Co MO (60 ng) and Wip1 MO (60 ng).

(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.

(F) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.

(G) Diagram of the post-translational Smad4 modifications by modifying enzymes and their downstream functions. P, phosphorylation; Ub, monoubiquitination; Me, methylation.

Data of (A, B, E, F) are representative of two independent experiments. Data of (C, D) are representative of three independent experiments.
Wip1 regulates Smad4 phosphorylation and inhibits TGF-β signaling (2020)
Figure 7

Figure 7. Wip1 inhibits TGF-β signaling by dephosphorylating Smad4 in its linker region.

(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm.

(K) Quantification of the results from in situ hybridization analysis in (A-J).

(L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4.

(M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.

(N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm.

(O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.

(P) Proposed model of the mechanism by which Wip1 controls the transcriptional activity and stability of Smad4. See the discussion for details.

Expanded View Figure Legends
Nuclear envelope deformation controls cell cycle progression in response to mechanical force (2019)
Figure 1

Figure 1 - Nuclear flattening is necessary for G1 to S transition.

(a) Cell height was measured using AFM for HeLa-FUCCI in the indicated phase of mitosis. Below: representative images of synchronised HeLa-FUCCI (box plot, median min/max, n = 20 minimum,****, ####p<0,001 one-way ANOVA - Tukey's multiple comparisons post-test, scale bar 15 µm)

(b) Representative images of synchronised HeLa stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 15 µm).

(c) Nucleus height was measured by immunofluorescence using Hoechst staining (n = 29 minimum for nucleus height, *p<0.5, ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

(d) Representative images of synchronised MEFs stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 5 µm, bottom scale bar x= 5 µm z= 5 µm).

(e) Nucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m. (n = 29 minimum for nucleus height, ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

(f) Schematic representation of the method used to induce changes in nuclear shape independently of the actomyosin cytoskeleton. See Figure EV1 for more details. Briefly, cells are treated with DMSO (Control-Ctrl), blebbistatin only (Blebb) and blebbistatin whereas an agarose pad (AP) is used to flatten their nuclei and restore a nuclear morphology similar to those of control cells (Blebb + AP). XZ view of representative nuclei (Lamin-A/C) are shown in these three conditions (scale bar x= 5 µm z= 5 µm).

(g) Nucleus height was measured by immunofluorescence using lamin A/C staining. Data are presented as mean ± s.e.m. (n = 20 for nucleus height, ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

(h) Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.

(i) Corresponding percentage of HeLa-FUCCI in mitosis. Data are presented as mean ± s.e.m. (n = 24 fields minimum from 4 independent experiments, ***p<0.01 one-way ANOVA - Tukey's multiple comparisons post-test).

(j) Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions.

(k) Corresponding percentage of EdU positive cells. Data are presented as mean ± s.e.m. (n = 4 independent experiments with at least 60000 events for each condition per experiment. ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).
Nuclear envelope deformation controls cell cycle progression in response to mechanical force (2019)
Figure 2

Figure 2 - Nuclear shape regulates transcription factor activity.

(a) Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).

(b) TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels. Data are presented as mean ± s.e.m. (n = 4 minimum, t-test *p<0.5 **p<0.05 ***p<0.01 ****p<0.001).

(c) TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5). Data are presented as mean ± s.e.m.

(d) AP1 activity was assessed in the indicated conditions using AP1 reporter assay. Data are presented as mean ± s.e.m. (n = 5, t-test ****p<0.001).

(e) Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.
Nuclear envelope deformation controls cell cycle progression in response to mechanical force (2019)
Figure 3

Figure 3 - Nuclear flattening is sufficient to activate c-Jun and YAP independently of actomyosin contractility and cell spreading area.

(a) Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining.

(b) Corresponding quantifications of p-Jun Ser63 nuclear intensity. Data are presented as mean ± s.e.m. (n = 59 minimum from 2 independent experiments, ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

(c) Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining.

(d) Corresponding quantifications of YAP activity (ratio nucleus/cytosol). Data are presented as mean ± s.e.m. (n = 82 minimum from 4 independent experiments, ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

(e) Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP. Data are presented as mean ± s.e.m. (n = 19 minimum, ****p<0.001 Tukey's multiple comparisons test).

(f) Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition. Data are presented as mean ± s.e.m. (n = 32 minimum from two independent experiments, ****p<0.001 Tukey's multiple comparisons test).

(g) Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2. Data are presented as mean ± s.e.m. (n = 28 minimum, t-test, ****p<0.001).

(h) Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining.

(i) Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity. Data are presented as mean ± s.e.m. (n = 15 minimum ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).
Nuclear envelope deformation controls cell cycle progression in response to mechanical force (2019)
Figure 4

Figure 4 - TEAD and AP1 activation are distinctively controlled by nuclear shape.

(a) Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio). Data are presented as mean ± s.e.m. (n = 59 minimum from 3 independent experiments, **p<0.05 ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

(b) Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.

(c) Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Data are presented as mean ± s.e.m. (n = 8, *p<0.5 ***p<0.01 one-way ANOVA - Tukey's multiple comparisons post-test).

(d) Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH. Data are presented as mean ± s.e.m. (n = 6, *p<0.5 ***p<0.01 one-way ANOVA - Tukey's multiple comparisons post-test).

(e) Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor. Data are presented as mean ± s.e.m. (n = 18 minimum, ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test.).

(f) Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm.

(g) Corresponding quantification of p-Jun Ser63 nuclear intensity. Data are presented as mean ± s.e.m. (n=49 minimum, **p<0.05 ***p<0.01 ****p<0.001 one-way ANOVA - Tukey's multiple comparisons post-test).

Expanded View Figures
CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels (2016)
Figure 1

Figure 1. Creation of Clpp knockout mice

(A) Targeting strategy for the conditional disruption of the Clpp gene. Map of the genomic locus region of the wild type Clpp gene (wild type), the Targeting Vector, the Clpp locus after homologues recombination (Targeted Allele) and targeted allele after Flp-recombinase-mediated excision of the PuroR cassette (Conditional KO Allele- ClpploxP) and the knocked-out allele after Cre-recombinase-mediated excision of the exons 3-5 (Constitutive KO Allele – Clpp-). The position of primers used for genotyping is indicated by red arrows.

(B) Southern Blot analysis of targeted ES cells. Positions of wild type (WT - 14.7kb) and targeted (loxP - 12.8kb) alleles are indicated.

(C) Genotyping analysis of wild type (Clpp+/+), heterozygous (Clpp+/-) and homozygous (Clpp-/-) Clpp knockout animals. DNA fragments corresponding to the wild type (WT-199 bp), and knockout (KO - 273 bp) Clpp loci are indicated.

(D) Analysis of Clpp transcript levels in heart (n=4). Bars represent mean ± standard deviation (SD). Student’s t-test was used to determine the level of statistical difference.

(E) Western blot analysis of CLPP protein levels in heart (He), quadriceps (Quad), liver (Li), kidney (Ki), lung (Lu), brain (Br), spleen (Sp) and pancreas (Pa). TUBULIN and ACTIN are used as a loading control. The arrowhead indicates positions of CLPP, and the star indicates position of cross-reacting bands.
CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels (2016)
Figure 2

Figure 2. Mitochondrial function in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) heart mitochondria

(A) Volcano plot of proteomic data reveals significant changes in proteins of the respiratory chain and factors involved in mitochondrial protein synthesis (FDR < 0.05).

(B) Bar chart representing enriched Gene Ontology (GO) terms. FDR was calculated by Benjamini-Hochberg correction (n=4).

(C) Respiratory chain enzyme activities. Relative enzyme activities of respiratory chain enzyme C I (NADH coenzyme Q reductase), C I/III (NADH cytochrome c reductase), C II (succinate dehydrogenase), C II/III (succinate cytochrome c reductase) and C IV (cytochrome c oxidase) in mitochondrial extracts from wild type and Clpp knockout hearts. (n=5). Bars present mean levels ± SD. Statistical difference was calculated by Student’s t-test and changes are presented in the figure (n=5).

(D) Substrate-dependent oxygen consumption rates in isolated heart mitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 – ADP stimulated oxygen consumption rate; State 4 – respiration rate after addition of oligomycin; Uncoupled – oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student’s t-test was used to determine the level of statistical difference (n=7).

(E) Decrease of steady-state levels of respiratory chain subunits in Clpp-knockout hearts relative to wild type (set to 1). VDAC or TOMM20 were used as loading controls. Bars present mean levels ± SD. Student’s t-test was used to determine the level of statistical difference (n=4).

(F) Blue Native Page Analysis (BN-PAGE) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I (anti-NDUFA9); C III (anti-UQCRC1) and C IV (anti-COX4A).
CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels (2016)
Figure 3

Fig. 3. MtDNA and mtRNA levels in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) hearts.

(A) Quantification of mtDNA levels after Southern blot analysis. Bars present mean levels ± SD (n=4).

(B-C) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4).

(D) Western blot analysis of LRPPRC levels. VDAC was used as a loading control.

(E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.
CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels (2016)
Figure 4

Fig. 4. Analysis of mitochondrial protein synthesis and mitoribosome assembly in wild type (Clpp+/+) and Clpp knockout (Clpp-/-) heart mitochondria

(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated.

(B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated.

(C) Quantitative analysis of the COXI recovery upon treatment with antibiotics (Actinonin, DOXY – doxycycline and CAP – chloramphenicol) as in B. The values represent ratios of recovered (48h, - antibiotics) versus untreated (0h) COXI levels. Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4). (Actinonine and CAP n=3, DOXY n=4).

(D) Sucrose density gradient sedimentation profile of ribosomal proteins and mtRNAs from heart mitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunobloting with antibodies against MRPS35 and MRPL12. The profile of a given mt-RNA is calculated after qRT-PCR analysis of individual fractions and corrected for its abundance in the cell according to results of Northern blot analysis. Results of individual experiments are provided for 12S and 16S rRNA, and for mt-mRNAs, results of distribution from two independent experiments using probes against ATP6, COXIII and ND5 were combined. Error bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (n=3).

(E) Relative association of 12S rRNA, 16S rRNA and mt-mRNAs with mitoribosomes (55S) in Clpp knockout mitochondria comparing to wild type. Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (12S and 16S n=3; mRNA n=6).
CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels (2016)
Figure 5

Fig. 5. Analysis of potential CLPP substrates involved in mitochondrial translation

(A) Network analysis of 66 potential CLPP substrates using only high confidence interactions (> 0.7). Known ClpXP substrates are shown in boxes with a green frame. Respiratory chain proteins and ribonucleoproteins are indicated in dark grey and orange, respectively.

(B) Western blot analysis and subsequent quantification of ERAL1 and EFG1 levels from Clpp knockout (Clpp-/-) heart mitochondria relative to wild type (Clpp+/+). Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (n=4). The arrowhead indicates positions of EFG1.

(C) Expression levels of potential CLPP substrates from Clpp knockout (Clpp-/-) heart mitochondria relative to wild type (set to 1). Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (n=4).

(D) Cycloheximide (CHX) chase assay to determine the stability of ERAL1 and EFG1. Proteins are visualized by Western blot with respective antibodies. Proteins are isolated after 2, 4, 6, 8,10 and 12 hours after beginning of chase (0). The arrowhead indicates positions of ERAL1.

(E) Western blot analysis and subsequent quantification of protein levels in wild type (C1, C2) and Clpp knockout (KO1, KO2) MEFs upon Clpx siRNA (Clpx) relative to untreated (Ctrl) wild type. Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (n=6). The arrowhead indicates positions of either CLPX or ERAL1, and stars indicate position of cross-reacting bands.
CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels (2016)
Figure 6

Fig. 6. Association of potential candidates with small ribosomal subunits

(A) Sucrose density gradient sedimentation profile (“short gradients”) of ribosomal proteins and transcripts from control (Clpp+/+) and Clpp knockout (Clpp-/-) heart mitochondria. The migration of ribosomal subunits (28S and 39S) and mitoribosome (55S) was detected by immunoblotting with antibodies against MRPS35 and MRPL12. 12S rRNA was analysed by Northern blot.

(B) Sedimentation profiles of ERAL1, MRPS28, EFG1, CLPP, CLPX, EFTU and MTIF2 in heart mitochondria as in A.

(C) Relative abundance of proteins bound to the 28S ribosomal subunit (fractions 3 and up). Bars represent mean ± SD. Student’s t-test was used to determine the level of statistical difference (ERAL1 n=4; EFG1, CLPX, MTIF2 n=3; EFTU n=2).

(D) Sucrose density gradient sedimentation profile (“long gradients” as in figure 4D) of ERAL1, MRPS35, MRPL37, CLPX and EFG1 from control (Clpp+/+) and Clpp knockout (Clpp-/-) heart mitochondria. The same amount of protein lysates were separated by the gradient and the amount of each individual protein is estimated after Western blot analysis (Input). Only fractions 4 - 11 containing the 28S, 39S and 55S ribosomal fractions were blotted to avoid high signals arising in free fractions (1-3).