Journal: Nature cell biology

Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

doi: 10.1038/ncb3592

Figure Lengend Snippet: ( a,b ) FRET analysis of Cdc42 activity at the apical aspect of MDCK cells conditionally expressing Dbl3-myc. ( c,d ) Confocal microscopy of pMLC and F-actin at the apical membrane domain of MDCK cells conditionally expressing Dbl3-myc induced with tetracycline in the absence or presence of Cdc42 inhibitor. White arrowheads highlight the apical membrane cortex labelled for F-actin. ( e,f ) Analysis of aPKCζ localization in MDCK cells with tetracycline-inducible Dbl3-myc expression. White arrowheads point to the lateral junctional complex. ( g ) Schematic diagram illustrating activation of two effector mechanisms, MRCK/Myosin-II and Par6-aPKC by Dbl3 stimulated apical Cd42-activation. Quantifications show shown are (b) box blots (25th to 75th percentiles, with a line at the median; whiskers extend to the max/min data points; n=36 cells -Tet and n=46 cells +Tet; p value was calculated with a Wilcoxon test), or (d, f) based on n=3 independent experiments and showing the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Scale bars: 10 μm.

Article Snippet: Blebbistatin (10μM final concentration), the Cdc42 inhibitor ML141 (10μM), and the ROCKI/II inhibitor GSK 269962 (10nM) were purchased from Tocris Bioscience.

Techniques: Activity Assay, Expressing, Confocal Microscopy, Membrane, Activation Assay, Derivative Assay

Journal: Nature cell biology

Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

doi: 10.1038/ncb3592

Figure Lengend Snippet: ( a,b ) Levels of EGFP-Lifeact (EGFP-LA), Par polarity and brush border proteins localized at the apical membrane domain in MDCK cells conditionally expressing Dbl3-myc, treated with blebbistatin and followed by washout (WO). ( c,d ) Levels of EGFP-Lifeact (EGFP-LA) enrichment at the apical membrane domain of MDCK cells conditionally expressing Dbl3-myc following blebbistatin treatment and washout in the absence or presence of BDP5290 to inhibit catalytic activity of MRCK. ( e-i ) Measurements of co-localization coefficients of PAR polarity and brush border proteins at the apical membrane domain in MDCK cells conditionally expressing Dbl3-myc transiently inhibited with blebbistatin followed by wash out for 2 hours (green indicates co-localisation, see also and ; arrowheads point to apical membrane). ( j ) Schematic model of polarity induced by apical stimulation of Cdc42, activating a dual effector mechanism to generate asymmetric actomyosin contractions, apical PAR domain formation and membrane morphogenesis. Bright green represents co-localization of Par and brush border proteins. ( k-n ) Quantification of confocal sections of pupal retinas stained for the apical markers aPKC and Moesin comparing wild type cells and gek mutant cells ( k, l ), or wild type cells and cells mutant for gek expressing Sqh EE ( m, n ). Quantifications in panels b, d, f, h, and i are based on n=3 independent experiments and shown are the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Quantifications in panels k-n of protein expression levels compare measurements from wild type and neighbouring mutant cells (paired within sections; see for representative images); k, n=10 animals; l, n=8 animals; m and n, n=7 animals; p values were calculated with t-tests). Scale bars: 10 μm.

Article Snippet: Blebbistatin (10μM final concentration), the Cdc42 inhibitor ML141 (10μM), and the ROCKI/II inhibitor GSK 269962 (10nM) were purchased from Tocris Bioscience.

Techniques: Membrane, Expressing, Activity Assay, Staining, Mutagenesis, Derivative Assay

Journal: Nature Communications

Article Title: CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

doi: 10.1038/s41467-023-36398-z

Figure Lengend Snippet: a Schematic of macropinocytosis and the Clathrin Independent Carrier (CLIC) pathways, their effectors and inhibitors. b GII.4 replication in the presence of EIPA (Na + /H + exchanger inhibitor), ML141 (Cdc42 inhibitor), Wiskostatin (N-WASP inhibitor), NSC23766 (RAC1 inhibitor), CT04 (RhoI inhibitor), Blebbistatin (myosin inhibitor), and LY29402 (PI3K inhibitor) at 1 h (gray) and 24 h (orange). c GII.4 replication in the presence of Arf1 inhibitor Golgicide A (GCA at 1 h (black) and 24 h (blue). d GII.4-induced tubular carriers at 1 h (37 o C) detected using guinea pig anti-Sydney VLP polyclonal antibody (Gp Syd-pAb) for viral capsid (green) and phalloidin for actin (red). Images were taken using a ZEISS Laser Scanning Microscope LSM 980 with Airyscan 2. e Electron microscopy to identify CLIC structures in GII.4 VLP-treated HIEs. (1–7 structures/cell) compared to, mock-treated cells (no structures in n = 25 cells). f Confocal microscopy to detect GII.4 VP1 capsid (green) colocalization with gal-3 (red) on the cell surface at 10 min and 1 h (37 o C) after VLP treatment using anti-gal-3 and Gp Syd-pAb (n = 3 HIE replicates). g Effect of blocking GII.4 virus-galectin-3 (gal-3) surface interaction using anti-gal-3 antibody on GII.4 replication at 1 h (black) and 24 h (red). h Dot blot analysis investigating GII.4 VLP interaction with purified gal-3. i Probing CLIC carriers utilized in HIEs for endocytosis with Alexa Fluor™ 594 conjugated cholera toxin B (CTxB) (red) and GII.4 VLPs (green) with similar cargoes marked by white arrows ( n = 2 HIE replicates). Inset: Co-occurrence of CTxB and GII.4 VLPs in similar cargos, scale = 5 µm. All the experiments were repeated independently three times with similar results. In b , c , and g viral GEs were quantified using n = 2 independent HIE replicates for the 1 h and n = 3 independent HIE replicates for 24 h with two technical replicates/sample. The error bars represent mean ± SD with P values calculated using one-way ANOVA, Dunnett’s multiple comparisons test with comparisons at 24 h relative to untreated control. Source data are provided as a Source Data file.

Article Snippet: ML141 , SCBT , sc-362768.

Techniques: Laser-Scanning Microscopy, Electron Microscopy, Confocal Microscopy, Blocking Assay, Virus, Dot Blot, Purification, Control

Journal: Nature Communications

Article Title: CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

doi: 10.1038/s41467-023-36398-z

Figure Lengend Snippet: List of inhibitors, additives and recombinant proteins

Article Snippet: ML141 , SCBT , sc-362768.

Techniques: Recombinant