EMBO/soda-nel huggingface dataset:
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 5

Figure 5 - Impaired ciliogenesis by depletion of lamin A/C may partially result from nesprin 2 suppression.

A An equal amount of whole cell lysates from RPE cells or those expressing shLamin A/C or luciferase (shLuc) was analyzed by the immunoblotting with antibodies as indicated.

B-D RPE cells were infected with lentiviruses expressing shRNAs to nesprin 2 (shNesprine 2, clone #1 and #2) or luciferase (shLuc) as a control. An equal amount of whole cell lysates was analyzed by immunoblotting with antibodies as indicated (B). The cells were serum-starved for 48 h and then stained for acetylated tubulin (white), pericentrin (green), F-actin (red) and DNA (blue). The representative images are shown (C) The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The numbers of F-actin stress fibers within 60 μm2 around the basal bodies (as illustrated by the circle) were measured (D, n ≥ 53).

E,F RPE cells expressing shNesprine 2 or shLuc were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The percentage of the cells with cilia in the total counted cells (E, n ≥ 197) and the length of cilia (F, n ≥ 52) were measured.

Data information: In D and E, values (means ± s.d.) are from three independent experiments. In F, values (means ± s.e.m.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 6

Figure 6 - Deficiency in ciliogenesis by depletion of lamin A/C or nesprin 2 may be caused by disruption of the actin homeostasis.

A,B RPE cells expressing shRNAs specific to lamin A/C or nesprin 2 (shNesprin 2, clones #1 and #2) were stained for G-actin with DNase I (green). Representative images are shown (A). Scale bars, 20 μm. The proportion of nuclear G-actin fluorescence intensity to the whole cell was measured (B, n ≥ 67).

C An equal amount of whole cell lysates from RPE cells (control) and those expressing shRNAs to importin 9 (shImportin 9, clones #1 and #2) was analyzed by immunoblotting with antibodies as indicated.

D-F The cells expressing shRNAs to importin 9 were stained for G-actin (green) and F-actin (red). The representative images are shown (D). Scale bars, 20 μm or 10 μm (insets). The proportion of nuclear G-actin intensity to the whole cell (E, n > 152) and the cell spreading area (F, n ≥ 139) were measured.

G-I RPE cells and those expressing shRNAs to importin 9 were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h and then stained for acetylated tubulin (white), pericentrin (green) and F-actin (red). Representative images are shown (G). The left insets show the merged signals of acetylated tubulin and pericentrin. Scale bars, 20 μm or 2 μm (magnified images). The percentage of the cells with cilia (H, n ≥ 385) and the length of cilia (I, n ≥ 111) were measured.

Data information: In B, E and H, values (means ± s.d.) are from three independent experiments. In F and I, values (means ± s.e.m.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 7

Figure 7 - Depletion of lamin A/C or nesprin 2 causes a defect in docking of Arp2 and myosin Va-mediated preciliary vesicles to the basal body.

A RPE cells expressing shRNAs specific to luciferase (shLuc.), lamin A/C, or nesprin 2 were grown in the growth medium (+ serum) or serum-starved for 1 h (- serum) and then stained for Arp2 (red), CEP164 (a marker for the mother centriole, green) and acetylated tubulin (white). The representative images are shown. Note that Arp2 was found to localize exclusively at the mother centriole (upper panels) or at the both mother and daughter centrioles (lower panels). Scale bars, 0.5 μm. The percentage of cells with Arp2 localized exclusively at the mother centriole in the total counted cells was measured (n=131-240 cells in each group).

B The cells were serum-starved for 48 h and then stained for acetylated tubulin (green), Arp2 (red), and γ-tubulin (white). The representative images are shown. Scale bars, 1 μm (upper panels) and 0.5 μm (lower panels). Note that the cell with Arp2 at the both mother and daughter centrioles fails to form cilium.

C The cells were serum-starved for 48 h and then stained for myosin Va (red), acetylated tubulin (green) and γ-tubulin (white). Scale bars, 0.5 μm. The cartoon illustrates that myosin Va-associated vesicles (CV+PCV) specifically accumulate at the mother centriole in most control cells (as in the "one centriole" image). CV, ciliary vesicle. PCV, periciliary vesicle. The percentage of cells with myosin Va exclusively at the mother centriole in the total counted cells was measured (n=160-200 cells in each group).

Data information: In A and C, values (means ± s.d.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t-test.
Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis (2020)
Figure 8

Figure 8 - Depletion of lamin A/C suppresses the recruitment of TTBK2 to the mother centriole.

A RPE cells expressing shRNAs specific to luciferase (shLuc) or lamin A/C (shLamin A/C) were serum-starved for 48 h and then stained for acetylated tubulin (Ac-tubulin, green) and CEP164 (red). The representative images show that the fluorescence intensity of CEP164 at the mother centriole of cilia-negative (left column) and cilia-positive (right column) basal bodies are similar. The graphs shown are the quantification of CEP164 at cilia-negative basal bodies and cilia-positive basal bodies.

B The cells were serum-starved for 32 h and then treated with (+) or without (-) Cyto D for another 16 h. The cells were stained for acetylated tubulin (Ac-tubulin, green) and TTBK2 (red). The graphs shown are the quantifications of TTBK2 intensity at cilia-negative basal bodies and cilia-positive basal bodies.

C The cells as in (B) were stained for acetylated tubulin (Ac-tubulin, green) and CP110 (red). The representative images show that CP110 is not removed from the mother centriole at some of cilia-negative basal bodies (left column), whereas it is removed from the mother centriole at cilia-positive basal bodies (right column). The graphs shown are the quantifications of CP110-removal at cilia-negative basal bodies and cilia-positive basal bodies.

Data information: Scale bars, 0.5 μm (A-C, panels from cilia-negative basal body); 1 μm (A-C, panels from cilia-positive basal body). In A and C, values (mean ± s.d.) are from three independent experiments. In B, values (means ± s.e.m.) are from three independent experiments. n ≥ 60 in each group. Statistical significance of differences is assessed with a Student's t-test.

Expanded View Figure Legends
Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo (2022)
Figure 1

Figure 1: Nucleation of K8 filaments in E7.5 mouse embryo

A. Bright-field picture and scheme of an E7.5 Late Bud (LB) embryo showing germ layers in Embryonic (E) and Extra-Embryonic (ExE) regions (Left). Zoom on ExE region with annotations of ExE structures (Right).

B. Bright-field picture of a 0B K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=3).

C. Bright-field picture of EB K8-eYFP embryo (Left) and time series of two-photon live imaging of ExE region (Right). Time interval is 5 min (n=4).

D. Bright-field pictures of K8-eYFP LB embryos (n=30), and high magnification spectral acquisition of areas of ExE mesoderm corresponding to dashed white areas. K8-eYFP and DAPI appear in yellow and cyan, respectively.

Data information: Scale bars represent 25 µm, except for bright-field pictures in B-C where scale bars show 50 µm.
Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo (2022)
Figure 4

Figure 4: Loss of keratin results in defective ExE region expansion.

A. Z-projections of whole-mount C57BL/6J wild-type (WT, Left) and KtyII-/- (Right) embryos at LB stage stained for vimentin (green) and F-actin (Phalloidin, white).

Data information: Scale bars represent 25 µm.

B. Sagittal sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Krt8 (n= 15 and n=5, respectively), F-actin (n=11 and 2), Vimentin (n=5 and 1), E-cadherin (n=1 and 1), T (n=4 and 3), Dsg2 (n=15 and 5) and Pecam-1 (n=4 and 3). Data information: White arrows point toward allantois and scale bars represent 25 µm.

C. Scheme for measurements.

D-G. Quantification of embryo morphology at LB stage in C57BL/6J wild-type (white boxes/ black dots, n=22 including 16 in which allantois could be measured), and KtyII-/- embryos (grey boxes/ black triangles, n=9 including 8 in which allantois could be measured). (D) Embryonic region length. Ratios of whole embryo and ExE cavity lengths (E), allantois length and width (F) and amnion length and thickness (G), over embryonic region length. P-values were calculated using the Mann-Whitney-Wilcoxon test (ns: no statistical significance, *: p < 0.05, **: p < 0.005). Error bars represent SD.

H. Z-projections of C57BL/6J wild-type (Top) and KtyII-/- (Bottom) embryos at LB stage stained for F-actin (Phalloidin, white). Amnion is delimited by yellow dashed lines. Scale bar represents 25 µm.

I-I''. SEM images of opened C57BL/6J wild-type (n=3) and KtyII-/- (n=2) LB stage embryos with zooms on the allantois region (I'-I''). Scale bars represent 200 (I), 20 (I') and 10 (I'') µm.
Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo (2022)
Figure 5

Figure 5: Amnion single cell transcriptome

A. In Brachyury (T)-Cre; mTmG embryos, mesoderm-derived cells express membrane eGFP (mG, green) and the other cells membrane tdTomato (mT, grey).

B. Scheme of amnion isolation.

C. Uniform Manifold Approximation and Projection (UMAP) (Left, color-coded) and RNA velocity (Right, black arrows show streaming directions from unspliced to spliced RNA) of amnion cells where 6 unsupervised clusters were identified.

D. UMAP of eGFP, Krt8, Cdh1 and Cdh2.

E. EB K8-eYFP embryo stained for eYFP (yellow) and E-cadherin (white) (n=7) (Top). Dashed white area zoom on an amnion region which highlights the two cell layers (Bottom).

F. Dot plot of specific gene expression in amnion clusters.

G. Immunostaining for Cdx2 (n=20).

H. Immunostaining for Nrp1 (n=8) and Col1a1 (n=7), primarily expressed in Amnion Mesenchyme.

I. Immunostaining for Epcam (n=12), Cldn7 (n=7), and Lrp2 (n=6), primarily expressed in Amnion Ectoderm.

J-K. Sections of C57BL/6J wild-type and KtyII-/- E7.5 embryos immunostained for Nrp1 (K) (n=8 and 2, respectively), and Lrp2 (L) (n=3 and 2).

Data information: White arrows point toward signal in amnion. In (E) and (G-L), sagittal sections with anterior to the left. Scale bars represent 25 µm.
Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo (2022)
Figure 6

Figure 6: Chorion single cell transcriptome

A. Scheme of chorion isolation where mesoderm is in green (mG), and the other cells are in grey (mT).

B. UMAP (Left) and RNA velocity (Right) of chorion cells where 5 unsupervised clusters were identified.

C. UMAP of eGFP, Krt8, Cdh1 and Cdh2.

D. Dot plot of specific gene expression in chorion clusters.

E. Immunostaining for Cdx2 (n=20).

F. Immunostaining for DAPI (cyan, left), Sox2 (middle), and Gata2 (right). Sox2 is primarily expressed in Trophoblast Progenitors and Gata2 in Chorion Ectoderm.

G. Immunostaining for Cebpb, detectable in most cells with variable intensity.

H. Immunostaining for Fn1 (n=22), Dlk1 (n=8), and Acta2 (n=12), primarily expressed in Chorion Mesenchyme.

I. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Dlk-1 (n=8 and 4, respectively) and Acta2 (n=6 and 3).

Data information: F and G are adjacent sections from the same embryo (n=4). White arrows point toward the signal in chorion. Sagittal sections with anterior to the left. Scale bars represent 25 µm.
Keratin filaments mediate the expansion of extra-embryonic membranes in the post-gastrulation mouse embryo (2022)
Figure 7

Figure 7: Allantois single cell transcriptome

A. Scheme of allantois isolation where mesoderm is in green (mG), and the other cells are in grey (mT).

B. UMAP (Left) and RNA velocity (Right) of allantois cells where 6 unsupervised clusters were identified.

C. UMAP of eGFP, Krt8, Cdh1, and Cdh2.

D. Dot plot of specific gene expression in allantois clusters.

E. Immunostaining for T (n=5) (Primitive Streak and Allantois Base).

F. Immunostaining for Ifitm3 (n=7) (Allantois Base).

G. Immunostaining for Cdh11 (n=9), Stard8 (n=4) and Nrp1 (n=8) (Allantois Cortex).

H. Immunostaining for Pitx1 (n=4) (Allantois Mesenchyme).

I. Hhex-GFP (n=5) and immunostaining for Kdr (n=8) (Endothelial Progenitors).

J. Immunostaining for Runx1 (n=3) (Erythroid Progenitors).

K-L. Sections from C57BL/6J wild-type and KtyII-/- LB embryos immunostained for Cdh11 (Top) and Krt8 (Bottom) (K, n=8 and 4, respectively) and Ifitm3 (L, n=2 and 2). Data information: White arrows point toward signal in allantois. Sagittal sections with anterior to the left. Scale bars represent 25 µm.

EXPANDED VIEW FIGURE LEGENDS
ZAK-beta is activated by cell compression and mediates contraction-induced MAPK signaling in muscle (2022)
Figure 1

Figure 1 - Osmotic shock-induced activation of p38 and JNK requires both ZAK isoforms.

Gene-trap-based genetic screen in haploid human cells for regulators of p38 kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38 population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).

Human U2OS cells and cells deleted for ZAK (∆ZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting as indicated.

Schematic of ZAK protein isoforms. LZ, Leucine Zipper; SAM, Sterile Alpha-Motif; S, Sensor domain; CTD, C-Terminal Domain; SFBD, Stress Fiber-Binding Domain.

Unique gene-trap insertions (dots) mapped to the genomic ZAK locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38 activation (Sorbitol (Fig 1A) and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).

U2OS/S-HA-ZAKβ cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.

U2OS cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ were pre-treated with ZAK inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown material was used in a kinase assay, separated by SDS-PAGE and analyzed by autoradiography.

U2OS and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.

Osmotic shock activates p38 both via a ZAKα-dependent ribotoxic stress response and a previously uncharacterized ZAKβ-dependent stress response.

U2OS and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies. T1, TAK1; M2, MEKK2.
ZAK-beta is activated by cell compression and mediates contraction-induced MAPK signaling in muscle (2022)
Figure 2

Figure 2 - ZAKβ redistributes to nuclear domains and stress fibers after osmotic shock.

U2OS cells stably expressing Strep-HA-ZAKβ or -ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody and counter-stained with DAPI.

U2OS cells stably expressing ZAKβ-GFP were treated with sorbitol (500 mM, 1 h), pre-extracted, fixed and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line.

As in (B) except that Hela cells were transfected with ZAKbeta-GFP and immunostained with 53BP1 antibody.

Cells from (B) were imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame.

As in (D), except that cells were co-transfected with mCherry-FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers.

Schematic of GFP-tagged ZAKβ truncation constructs.

U2OS cells were transfected with constructs from (F) and imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame.

U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.

Data information: All scale bars, 10 μm.
ZAK-beta is activated by cell compression and mediates contraction-induced MAPK signaling in muscle (2022)
Figure 3

Figure 3 - ZAKβ responds to cellular compression but not stretching.

Schematic of mechanical perturbation of cells by compression and stretching.

U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.

U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).

U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).

U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).

Model of MAP3Ks involved in p38- and JNK-activating signaling in mechanically stimulated cells. ZAKβ is activated upon compressive load (left), in a manner dependent on stress fiber binding. TAK1 is activated by cell stretching (right).

Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.

Murine C2C12 myoblasts can be differentiated into myotubes by incubation in myotube differentiation (diff.) medium for 6 days.

C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.

C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.
ZAK-beta is activated by cell compression and mediates contraction-induced MAPK signaling in muscle (2022)
Figure 4

Figure 4 - Z-disc localized ZAKβ is activated by muscle contraction.

Genomic location of guide-RNA sequence (blue) and a derived knockout allele in exon 2 of the murine ZAK gene. PAM, Protospacer Adjacent Motif.

Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.

Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.

Schematic of in situ muscle contraction experiments. Mice were anaesthetized and one of the lower hindlimbs were subjected to electrically stimulated contraction (10 min.). Upon euthanization, tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were isolated. Tissue homogenates were processed for immunoblotting (e) or proteins were subjected to trypsin digestion, phospho-peptide enrichment and label-free quantification by mass spectrometry.

16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.

ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).

TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow.

C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1.

Schematic of electroporation rescue experiments. TA muscles were electroporated with GFP-ZAKβ constructs. After 3 days the muscles were exposed to in situ contraction and processed for immunoblotting.

16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.

Data information: All scale bars, 20 μm.
ZAK-beta is activated by cell compression and mediates contraction-induced MAPK signaling in muscle (2022)
Figure 5

Figure 5 - ZAKβ guards against skeletal muscle pathology in mice.

H&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm.

Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.

Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm.

Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.

Cross-sectional area of type I and type IIa fibers in soleus from 8-week-old WT and ZAK-/- male mice. Values indicate the mean from one specimen and error bars represent the standard deviation (n=3 biological replicates). *, p<0.05 in 2-way ANOVA corrected for multiple comparison using FDR (Benjamini, Krieger and Yekutieli).

Schematic of muscle transcriptomic analysis. Soleus and TA muscles were isolated from 16-18-week-old WT and ZAK-/- female mice (n=4 biological replicates). Purified RNA was subjected to polyA capture and deep sequencing.

Principal component analysis of the data obtained from (F).

Venn diagram from (F) of the overlap of differentially expressed genes (DEG) in soleus (Sol, blue) and TA (brown).

Mechanical perturbation of cells by osmotic shock, compression and muscle contraction activates ZAKβ and downstream kinases p38 and JNK. These reactions are dependent on sensing of stress fiber / Z-disc deformation by the ZAKβ SFBD domain.

Expanded View Figure legends