Journal: eLife

Article Title: Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration

doi: 10.7554/eLife.45853

Figure Lengend Snippet: List of qPCR primers.

Article Snippet: Commercial assay or kit , Cdc42 Pull-down Activation Assay Biochem Kit , Cytoskeleton , Cat#: BK034 , .

Techniques: Sequencing

Journal: eLife

Article Title: Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration

doi: 10.7554/eLife.45853

Figure Lengend Snippet: ( A ) Angular histograms showing the distribution of polarization angles from siControl (n = 11 images, from six independent experiments) and siROR2 (n = 11 images, from six independent experiments) transfected cells. ( B ) Polarity index box plots from siControl (n = 11 images, from six independent experiments), and siROR2 (n = 11 images, from six independent experiments) cells. p-values from unpaired t-test. ( C ) Co-localization (%) between Vinculin/VE-Cadherin in siControl and siROR2 transfected cells (n = 9 images, from three independent experiments). Data are mean ± SD, p-values from unpaired t-test. ( D ) Angular histograms showing the distribution of polarization angles of followers from wild type cells treated with either DMSO, iJNK (SP600125), iRac (NSC27632) or iCdc42 (ML141). n = 4 images, from two independent experiments. ( E ) Angular histograms showing the distribution of polarization angles from siControl (n = 14 images, from five independent experiments) and siCdc42 (n = 11 images, from five independent experiments) transfected cells. ( F ) Polarity index box plots from siControl (n = 14 images, from five independent experiments) and siCdc42 (n = 11 images, from five independent experiments) transfected cells. p-values from unpaired t-test. ( G ) Co-localization (%) between vinculin-VE-cadherin (n = 8 images, from three independent experiments) in siControl and siCdc42 transfected cells. Data are mean ± SD, p-values from unpaired t-test. ( H ) Detail of adherens junctions showing the association of actin stress fibers (phalloidin) to the adherens junctions (VE-Cadherin) of adjacent HUVECs in siCdc42 transfected cells. Nucleus labeled with Dapi. Scale bar, 20 µm. ( I ) Quantification of cell perimeter (%) composed of linear (blue), serrated (red) and reticular (green) in siControl and siCdc42 transfected cells (n = 78 and 75 cells, respectively, from two independent experiments). Data are mean ± SEM and p-values from unpaired t-test. ( J ) Quantification of the number of actin stress fibers connected to VE-cadherin positive cell-cell junctions in siControl or siCdc42 treated cells. N = 7 images, from three independent experiments. Data are mean ± SD, and p-values from unpaired t-test. ( K ) Pulldown of active GTP-bound Cdc42 in siControl and siROR2 transfected cells unstimulated or stimulated with recombinant human Wnt5a protein (rhWnt5a) (n = 1). ( L ) HUVEC expressing Cdc42-2G at adherens junction. Scale bar = 20 µm. ( M ) Box plots showing the number of Cdc42 FRET peaks per junction in siControl (n = 11 cell-cell interfaces, from two independent experiments) and siWNT5a (n = 9 cell-cell interfaces, from two independent experiments) transfected cells. p-values from unpaired t-test. ( N ) Box plots showing the number of Cdc42 FRET peaks per leading edge in siControl (n = 5 leading edges, from two independent experiments) and siWNT5a (n = 6 leading edges, from two independent experiments) transfected cells. p-values from unpaired t-test. ( O ) Left: example of a mouse retina sprouting front treated with PBS and Ml141 labeled for EC nuclei (Erg, green), lumen (Icam2, blue) and Golgi (Golph4, red). Right: higher magnification of the sprouting front showing high cell polarity coordination in PBS treated retinas and poor cell polarity coordination in Ml141 treated retinas. Scale bar, 200 µm. ( P ) Angular histograms showing the distribution of polarization angles of endothelial cells at the vascular sprouting front from mouse retinas treated with PBS (n = 4 retinas) or Ml141 (n = 5 retinas). ( Q ) Polarity index box plots of endothelial cells from mouse retinas treated with PBS (n = 4 retinas) or Ml141 (n = 5 retinas). p-values from unpaired t-test.

Article Snippet: Commercial assay or kit , Cdc42 Pull-down Activation Assay Biochem Kit , Cytoskeleton , Cat#: BK034 , .

Techniques: Transfection, Labeling, Recombinant, Expressing

Journal: eLife

Article Title: Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration

doi: 10.7554/eLife.45853

Figure Lengend Snippet: Working model for the role of non-canonical Wnt ligand WNT5a in mechanotransduction. Wnt5a, through ROR2, activates Cdc42 at adherens junctions, which is necessary for stable binding of vinculin to ɑ-catenin, and efficient mechanocoupling between endothelial cells. Low non-canonical Wnt signaling weakens adherens junctions, impairs force propagation, and disrupts collective cell migration of endothelial cells.

Article Snippet: Commercial assay or kit , Cdc42 Pull-down Activation Assay Biochem Kit , Cytoskeleton , Cat#: BK034 , .

Techniques: Binding Assay, Migration

Journal: eLife

Article Title: Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration

doi: 10.7554/eLife.45853

Figure Lengend Snippet: ( A ) Quantification of mRNA expression levels by qPCR showing the knockdown efficiencies of siRNAs against CDC42, CDH5, CTNNA1, FZD4, FZD6, FZD7, FZD8, ROR1, ROR2, RYK, VCL, WNT5a and WNT11. Data are mean ± SD, gene expression levels were normalized to GAPDH. ( B ) Western blot showing siRNA knockdown efficiency for α-Catenin (n = 2), VE-cadherin (n = 2), vinculin (n = 1) and Cdc42 (n = 1).

Article Snippet: Commercial assay or kit , Cdc42 Pull-down Activation Assay Biochem Kit , Cytoskeleton , Cat#: BK034 , .

Techniques: Expressing, Knockdown, Gene Expression, Western Blot

Journal: eLife

Article Title: Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration

doi: 10.7554/eLife.45853

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Cdc42 Pull-down Activation Assay Biochem Kit , Cytoskeleton , Cat#: BK034 , .

Techniques: Generated, Recombinant, Plasmid Preparation, Expressing, Mutagenesis, Clone Assay, Binding Assay, Sequencing, Bicinchoninic Acid Protein Assay, Activation Assay, In Situ, Western Blot, Purification, Molecular Weight, Marker, SYBR Green Assay, Software, Chemotaxis Assay, Migration, Immunostaining, Modification, Staining

Journal: bioRxiv

Article Title: Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

doi: 10.1101/2025.02.25.640099

Figure Lengend Snippet: (A) Bubble plot showing Reactome Pathway Analysis enrichment based on genes with significantly altered promoter accessibility in scATAC-seq data from control and p63-Ror2-KO cells. Pathways are ranked by the number of genes with altered promoter accessibility, represented by bubble size and red color gradients. (B) Quantitative RT-qPCR analysis of Rho GTPase-activating proteins Arhgap24 and Arhgap32 in FACS-isolated basal epithelial cells from control and p63-Ror2-KO mice. Gene expression levels were normalized to GAPDH, and fold changes were plotted relative to the control group ( p = 0.008 for Arhgap24 ; p < 0.001 for Arhgap32 ; n = 3 biological replicates per group). (C) Western blot for Ror2 showing efficient Ror2 knockdown with LeGO-shRor2 cells relative to LeGO-shLUC control cells. (D) Western blot analysis of GTP-bound RhoA, total RhoA, ROCK1, Cdc42, Rac1/2/3, and GAPDH in shLUC and shRor2 mammary basal epithelial cells with or without Wnt5a treatment. (E) Peak tracks illustrating accessible genomic elements for YAP1 downstream target genes Vgll3, Ctgf, and Ankrd1. These regions are less accessible in luminal cell types in p63-Ror2-KO groups. (F) Venn diagram showing the overlap between significantly altered transcription factors identified in scATAC-seq motif enrichment analysis and YAP1-binding transcription factors from previous ChIP sequencing data . (G) Western blot analysis of phospho-YAP1, total YAP1, TAZ, phospho-LATS1, LATS1, and GAPDH in shLUC and shRor2 mammary basal epithelial cells with or without Wnt5a treatment. (H) Quantitative RT-qPCR analysis of Hippo/YAP1 signaling genes in FACS-isolated basal epithelial cells from control and p63-Ror2-KO mice. Gene expression levels were normalized to GAPDH , and fold changes were plotted relative to the control group ( p = 0.015 for LATS2; p < 0.001 for other genes; n = 3 biological replicates per group). (I, J) Representative immunofluorescence images showing eGFP (green) and YAP1 (gray) in mammary gland cross-sections from (I) control and (J) p63-Ror2-KO mice at 4 weeks post-tamoxifen injection. Scale bars: 20 μm for merged images and 10 μm for magnified insets (I’, I”, J’, J”). White arrowheads indicate nuclear YAP1 in eGFP + basal cells, while yellow arrowheads indicate the absence of nuclear YAP1 in eGFP + luminal cells within p63-Ror2-KO mammary ducts.

Article Snippet: The following primary antibodies were used, with their respective dilutions indicated: Ror2 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), RhoA (1:1000; #2117; Cell Signaling Technology [CST], Danvers, MA), ROCK1 (1:1000; #4035; CST), Cdc42 (1:1000; #2466; CST), Rac1/2/3 (1:1000; #2465; CST), YAP/TAZ (1:1000; #8418; CST), phosphorylated YAP (p-YAP, 1:1000; #13008; CST), LATS1 (1:1000; #3477; CST), phosphorylated LATS1 (p-LATS1, 1:1000; #9157; CST), and GAPDH (1:2500; #5174; CST).

Techniques: Control, Quantitative RT-PCR, Isolation, Gene Expression, Western Blot, Knockdown, Binding Assay, ChIP-sequencing, Immunofluorescence, Injection

Journal: Cell Reports Medicine

Article Title: A Rac-specific competitive inhibitor of guanine nucleotide binding reduces metastasis in triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102233

Figure Lengend Snippet: A41 selectively impairs RAC protein activation (A) Representative surface plasmon resonance (SPR) sensograms of binding of immobilized RHOA or CDC42 with indicated increasing concentrations of A41 ( N > 3). (B) Effect of A41 on GEF-stimulated RAC2, RHOG, RHOA, and CDC42 nucleotide exchange. Purified small G proteins were pre-loaded with GDP and then nucleotide exchange was monitored by the increase in fluorescence following mant-GTP binding in the absence and presence of A41 (5 μM) ( N = 3). K obs is expressed as mean ± SEM. ∗ p < 0.001 vs. control. (C) Effect of A41 on RAC1 G30S activation. RAC1 G30S activation was monitored by the increase in fluorescence following mant-GTP binding in the absence and presence of A41 (5 μM). The GEF TRIO was used to induce nucleotide exchange ( N = 3). Data are expressed as mean ± SEM. (D) Immunoblot analysis and associated quantification of RAC-GTP level and total RAC expression in NIH-3T3 fibroblasts expressing RAC wild-type (RAC1 WT) or RAC1 oncomutants (RAC1 P29S and RAC1b) in the absence (Ctrl) and presence of A41 (10 μM). RAC activation was measured as the ratio of RAC-GTP to total RAC and expressed relative to Ctrl condition. Data are presented as mean ± SEM. ∗∗∗ p < 0.001 vs. controls.

Article Snippet: RAC1, RHOA and CDC42 purified proteins (respectively RH01, RC01 and CD01, Cytoskeleton) were diluted to 5 μg/mL in Na + acetate buffer (pH 5.0) and injected into sensor chip CM5 (GE Healthcare) in a Biacore T200 (GE Healthcare) that was activated with NHS/EDC buffer.

Techniques: Activation Assay, SPR Assay, Binding Assay, Purification, Fluorescence, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

doi: 10.1101/2023.10.27.563985

Figure Lengend Snippet: A. aPKC phosphorylates CDC-42 in vitro . Immunoblot showing that human Cdc42 can be phosphorylated by human aPKC (PKC ζ) in vitro using purified recombinant proteins incubated with ATP-gamma-S. After each kinase reaction, the products were alkylated, and thiophosphorylated Cdc42 (pCdc42) was detected with an anti-thiophosphate ester antibody (see ). Total Cdc42 detection is shown as loading control. B. Protein sequence conservation for Cdc42 and Rac1 (CED-10, C. elegans homologue) around serine 71 (red). Conserved arginines, which are part of aPKC consensus site are highlighted (green). C. Immunoblot showing the detection of phosphorylated CDC-42 and Par6 (positive control) from an in vitro kinase assay containing co-purified human PKCΙ - Par6 and wild-type C. elegans CDC-42 (WT) or CDC-42(S71A) (SA). Thiophosphorylation detection was done as in (A). In three independent blots we consistently observed more phosphorylation of CDC-42 wild type compared to CDC-42(S71A) (~17 times more, after loading correction and background subtraction, i.e. subtraction of signal detected in the corresponding minus ATPγS lane). A similar increase in phosphorylation was observed for Par6 in the CDC-42 kinase reaction compared to that of CDC-42(S71A). D. Representative midsection confocal images of live embryos at establishment, maintenance and 2-cell stage showing GFP::CDC-42, GFP::CDC-42(S71A) and GFP::CDC-42(S71E). Zygotes are always oriented anterior to the left. In these CDC-42 reporter lines we deplete endogenous CDC-42 by RNAi that targets its 3’ UTR, in this way preventing possible phenotypic rescue by endogenous CDC-42. E. CDC-42 anterior and posterior cortical intensities (values are normalised by its corresponding cytoplasmic CDC-42 levels) observed in CDC-42, CDC-42(S71A) and CDC-42(S71E) maintenance stage embryos (graph shows all data points and mean ± CI 95%). Note that CDC-42(S71E) presents a very weak cortical localisation with no asymmetry (no difference in intensity at anterior vs. posterior). F. CDC-42 intensity profiles spanning the anterior membrane of CDC-42, CDC-42(S71A) and CDC-42(S71E) maintenance stage zygotes, showing mean ± SD. Briefly, a 60x60 pixel area from a straightened anterior cortex (see inset in A in GFP::CDC-42 maintenance) was projected in the y-axis to give a cross section profile spanning the cortex/membrane. The values are normalised so that the cytoplasmic levels are set to 1. Number of embryos studied in D. and E.: 13 CDC-42, 16 CDC-42(S71A), and 16 CDC-42(S71E). G. A representative HILO image of wild-type CDC-42 at the membrane. Below we show representative tracks obtained by following CDC-42 particles at the membrane in the different CDC-42 variants. H. Graph showing the diffusion coefficients (D) of the mobile fraction of CDC-42 variants and PAR-6, obtained from gamma fits to all mobile tracked data (coloured lines, errors represent 95% confidence intervals on the fits). See . Diffusion coefficients of gamma fits for mobile data obtained from individual embryos is shown by the position of the grey circles (circle size represents the number of tracks analysed per embryo and the lines represent 95% confidence intervals on the fits). I. Proportion of tracks found in the immobile fraction (D < 0.1 μm 2 /s) in each condition (graph shows all data points and mean ± SEM). Horizontal black line indicates the limit of detection of immobile fraction (baseline, See ). Average number of tracks (± SEM) at maintenance stage for the embryos analysed in E and F, CDC-42: 3295 ± 57 tracks/embryo in 5 embryos; CDC-42(S71A): 3047 ± 62 in 6 embryos; CDC-42(S71E): 3532 ± 47 in 9 embryos and PAR-6: 1380 ± 790 in 4 embryos. Unpaired, two-tail Student’s t-test *p<0.05, ****p<0.0001, ns not significant. Scale bar in establishment zygote: 10 µm. See also - , and .

Article Snippet: In vitro kinase assays were performed in the presence or absence of different recombinant human atypical PKC (50 or 100 ng of PKC ζ, #14–525 Merk Millipore; 1µg of PKC I/αPar6 complex, gift from Shona Ellison, Mc Donald lab, Crick Institute) and with different recombinant Cdc42 (3µg of human Cdc42, #CD01 Cytoskeleton; 3µg of recombinant MBP tagged- C. elegans CDC-42 or CDC-42(S71A)), which were incubated for 1 hour at 30 °C in 30 µl kinase-assay buffer (25 mM Tris pH 7.5, 25 mM NaCl, 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT) containing 1mM ATP-gamma-S (# ab138911,Abcam).

Techniques: In Vitro, Western Blot, Purification, Recombinant, Incubation, Sequencing, Positive Control, Kinase Assay, Membrane, Diffusion-based Assay

Journal: bioRxiv

Article Title: Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

doi: 10.1101/2023.10.27.563985

Figure Lengend Snippet: A. Graphical representation of the experimental set up. Briefly, we use a membrane-tethered nanobody against GFP (PH-GBP) that can dock at the membrane GFP::CDC-42, GFP::CDC-42(S71A) or GFP::CDC-42(S71E). B. Zygotes with membrane-docked CDC-42 are immunostained for aPKC to determine aPKC membrane levels at the anterior domain (anterior-most selected region). C. Representative anterior membrane regions (three per condition) showing aPKC levels in the non-membrane docked GFP:CDC-42 reporter and in the membrane-docked GFP::CDC-42, GFP::CDC-42(S71A) and GFP::CDC-42(S71E) reporters (in all CDC-42 reporters endogenous CDC-42 is depleted). D. Average aPKC intensity values at the anterior membrane (measured in a 60x60 pixel area from a straightened anterior-most cortex) of zygotes during polarity maintenance. Anterior membrane intensity of aPKC for each zygote is normalised with its corresponding aPKC cytoplasmic level (graph shows all data points and mean ± CI 95%). Significant differences in the levels of anterior aPKC are observed for membrane docked GFP::CDC-42(S71A) and GFP::CDC-42(S71E) when compared to membrane-docked wild-type CDC-42. Number of embryos analysed in each condition are indicated in the graph. Unpaired, two-tail Student’s t-test **p<0.01, ***<0.001. See also - . E. Graphical representation of the lipid nanodisc single-cell pull-down (sc-SiMPull) experiment. Cells expressing GFP::CDC-42 variants and carrying endogenously tagged HaloTag::aPKC are lysed using a pulsed infrared laser in the presence of amphipathic polymers to form native lipid nanodiscs. sc-SiMPull was used to capture GFP::CDC-42 variants, which were detected using single-molecule TIRF microscopy. Complexes containing HaloTag::aPKC were identified and dissociation rate constants (k off ) of those complexes were measured (see and ). F. Dissociation rate constants measured for CDC-42/aPKC, CDC-42(S71A)/aPKC and CDC-42(S71E)/aPKC complexes extracted from late establishment and maintenance stage zygotes. Gray circles represent individual experiments, with the size of the circle indicating the number of CDC-42/aPKC complexes captured from each zygote and the error bars representing the Bayesian 95% credible interval for the estimated k off . Colored bars show the maximum probability estimate and Bayesian 95% credible intervals for the k off obtained by pooling all single-molecule measurements. n = 897 total CDC-42/aPKC complexes from 13 embryos (WT); n = 1,635 total CDC-42/aPKC complexes from 11 embryos (S71A); n = 676 total CDC-42/aPKC complexes from 5 embryos (S71E). Orange bars are measurements of k off for the complex of wild-type mNG::CDC-42 and PAR-6 during polarity establishment or polarity maintenance (Deutz et al., 2023), shown here for comparison. See also .

Article Snippet: In vitro kinase assays were performed in the presence or absence of different recombinant human atypical PKC (50 or 100 ng of PKC ζ, #14–525 Merk Millipore; 1µg of PKC I/αPar6 complex, gift from Shona Ellison, Mc Donald lab, Crick Institute) and with different recombinant Cdc42 (3µg of human Cdc42, #CD01 Cytoskeleton; 3µg of recombinant MBP tagged- C. elegans CDC-42 or CDC-42(S71A)), which were incubated for 1 hour at 30 °C in 30 µl kinase-assay buffer (25 mM Tris pH 7.5, 25 mM NaCl, 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT) containing 1mM ATP-gamma-S (# ab138911,Abcam).

Techniques: Membrane, Expressing, Microscopy, Comparison

Journal: bioRxiv

Article Title: Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

doi: 10.1101/2023.10.27.563985

Figure Lengend Snippet: A. PAR retraction difference assay showing zygote phenotypes that indicate an increase or decrease in the CDC-42/PAR-6/aPKC membrane domain that is independent from PAR-3. B. Representative flattened membranes from PAR-3 and aPKC co-immunostained zygotes, showing the difference in retraction observed between PAR-3 and aPKC in wild type, cdc-42 depleted (RNAi of its 3’ UTR), GFP::CDC-42, GFP::CDC-42(S71A) and GFP::CDC-42(S71E) strains (in all CDC-42 reporters endogenous CDC-42 is depleted). Four cortices are shown for each strain. PAR-3 and aPKC are shown for the first zygote in each strain. For the other zygotes only aPKC is shown but its position is relative to the end of its corresponding PAR-3 domain (dashed vertical line). C. Quantification of PAR retraction difference. S71A mutation in CDC-42 promotes the CDC-42/PAR-6/aPKC membrane domain (as shown by the larger expansion of aPKC relative to PAR-3), whereas S71E mutation decreases membrane CDC-42/PAR-6/aPKC. D. Representative cortical confocal images of PAR-3 and aPKC in the indicated CDC-42 strains. In cortical sections we have analysed aPKC and PAR-3 co-localisation ( E ), aPKC asymmetry using ASI index (normalised against wild-type asymmetry, see ) ( F ) and aPKC coefficient of variance as a means to measure clustered (closer to 1) vs. diffusive state (closer to 0) ( G ). All box plots show the median ± IQR and all data points. Number of embryos analysed indicated in the graphs. Unpaired, two-tail Student’s T test *p<0.05 **p<0.01, ***p<0.001, ****p<0.0001. Scale bar: 10 µm.

Article Snippet: In vitro kinase assays were performed in the presence or absence of different recombinant human atypical PKC (50 or 100 ng of PKC ζ, #14–525 Merk Millipore; 1µg of PKC I/αPar6 complex, gift from Shona Ellison, Mc Donald lab, Crick Institute) and with different recombinant Cdc42 (3µg of human Cdc42, #CD01 Cytoskeleton; 3µg of recombinant MBP tagged- C. elegans CDC-42 or CDC-42(S71A)), which were incubated for 1 hour at 30 °C in 30 µl kinase-assay buffer (25 mM Tris pH 7.5, 25 mM NaCl, 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT) containing 1mM ATP-gamma-S (# ab138911,Abcam).

Techniques: Membrane, Mutagenesis

Journal: bioRxiv

Article Title: Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

doi: 10.1101/2023.10.27.563985

Figure Lengend Snippet: A. Representative cortical confocal images of NMY-2 in establishment wild-type, cdc-42 depleted, CDC-42, CDC-42(S71A) and CDC-42(S71E) zygotes. In all CDC-42 reporter lines endogenous CDC-42 is depleted. B. Graph showing the coefficient of variation (CV) as a measurement of myosin cortex organisation. Well-defined foci lead to higher CV values, whereas a dispersed appearance leads to lower values . C. Representative time projections (100 frames) of NMY-2 confocal cortical images during actomyosin flow in polarity establishment zygotes, showing the actomyosin cortex clearance from the posterior in the different conditions. D. Comparison of average AP flow velocity in the posterior of embryos (representative area studied for AP flow measurements highlighted in wild type) for the specified conditions. E. Co-plotting of CV (higher to lower from left to right) and NMY-2 flow for ease of comparison. Showing median ± CI 95%. F. Representative cortical confocal images of NMY-2 in maintenance (prior to cleavage furrow) wild-type, cdc-42 depleted, CDC-42, CDC-42(S71A) and CDC-42(S71E) zygotes. In all CDC-42 reporter lines endogenous CDC-42 is depleted. G. NMY-2 intensity at the anterior half of the zygote normalised by its intensity at the posterior during polarity maintenance. All box plots show median ± IQR and all data points. Unpaired, two-tail Student’s t-test *p<0.05, ***p<0.001, ****p<0.0001, ns not significant. Scale bar: 10 µm.

Article Snippet: In vitro kinase assays were performed in the presence or absence of different recombinant human atypical PKC (50 or 100 ng of PKC ζ, #14–525 Merk Millipore; 1µg of PKC I/αPar6 complex, gift from Shona Ellison, Mc Donald lab, Crick Institute) and with different recombinant Cdc42 (3µg of human Cdc42, #CD01 Cytoskeleton; 3µg of recombinant MBP tagged- C. elegans CDC-42 or CDC-42(S71A)), which were incubated for 1 hour at 30 °C in 30 µl kinase-assay buffer (25 mM Tris pH 7.5, 25 mM NaCl, 5 mM MgCl2, 0.5 mM EGTA, 1 mM DTT) containing 1mM ATP-gamma-S (# ab138911,Abcam).

Techniques: Comparison