Journal: Nature structural & molecular biology

Article Title: Structurally encoded intraclass differences in EphA clusters drive distinct cell responses

doi: 10.1038/nsmb.2617

Figure Lengend Snippet: a) Schematic overview showing Eph domain composition: ligand-binding domain (LBD), sushi, epidermal-growth-factor-like (EGF), fibronectin type III (FN1 and FN2), transmembrane helix (TM), tyrosine kinase and sterile-alpha motif (SAM). Domains are coloured separately for the ectodomain. b) Rounding responses of non-transfected control and Eph-transfected HeLa cells upon ephrinA5-Fc stimulation were measured. Average adherent cell surface areas were normalized using the values at time =0 (before receptor stimulation). Statistical significance was determined using one-way ANOVA and Tukey’s post hoc test and is shown with red stars (control, EphA4) and black stars (EphA2, EphA4). Error bars denote s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. c) Hela cell stripe assay. Adhesion of Eph-transfected cells to ephrinA5-Fc coated surfaces is shown (50% corresponds to random distribution, > 50% reflects adhesion). Statistical significance was calculated with one-way ANOVA and Tukey’s post hoc test and is shown with red stars (to EphA4), blue stars (to EphA2) and grey stars (to control). Error bars denote s.e.m. ** P < 0.01.

Article Snippet: EphrinA5-Fc (R&D Cat. no. 374-EA) at 0.2 mg ml −1 in PBS was mixed with goat anti-human IgG Fcγ (Jackson ImmunoResearch) and imaging medium in a 5:1:28 ratio, and incubated for 2 hours at room temperature.

Techniques: Ligand Binding Assay, Transfection, Stripping Membranes

Journal: Nature structural & molecular biology

Article Title: Structurally encoded intraclass differences in EphA clusters drive distinct cell responses

doi: 10.1038/nsmb.2617

Figure Lengend Snippet:

Article Snippet: EphrinA5-Fc (R&D Cat. no. 374-EA) at 0.2 mg ml −1 in PBS was mixed with goat anti-human IgG Fcγ (Jackson ImmunoResearch) and imaging medium in a 5:1:28 ratio, and incubated for 2 hours at room temperature.

Techniques: Methylation

Journal: Nature structural & molecular biology

Article Title: Structurally encoded intraclass differences in EphA clusters drive distinct cell responses

doi: 10.1038/nsmb.2617

Figure Lengend Snippet: a) Cartoon diagrams are shown of complete EphA4 ectodomain (EphA4 ecto ), unliganded or in complex with ephrinA5 or ephrinB3 receptor-binding domain (RBD). EphA2 domains are coloured separately as indicated. Ligand-binding domain (LBD), epidermal-growth-factor-like (EGF), fibronectin type III domains 1 and 2 (FN1 and FN2). Ephrins are coloured in grey. b) Crystal lattice assemblies that are compatible with clustering of receptors on the same cell (in cis ) are shown as surface representations for the four crystal structures presented (side and top views). Colours are as in panel a.

Article Snippet: EphrinA5-Fc (R&D Cat. no. 374-EA) at 0.2 mg ml −1 in PBS was mixed with goat anti-human IgG Fcγ (Jackson ImmunoResearch) and imaging medium in a 5:1:28 ratio, and incubated for 2 hours at room temperature.

Techniques: Binding Assay, Ligand Binding Assay

Journal: Nature structural & molecular biology

Article Title: Structurally encoded intraclass differences in EphA clusters drive distinct cell responses

doi: 10.1038/nsmb.2617

Figure Lengend Snippet: a) Schematic representation of transmembrane Eph constructs: EphA2 blue, EphA4 red. Chimeric proteins were engineered by swapping ectodomains (A2A4 or A4A2). Stars mark point mutants in the HI-loop, sushi dimerization and major ephrin-binding sites. All constructs contain an N-terminal Flag tag and a C-terminal mVenus or mCherry tag. b) HeLa cell rounding responses measured 10 minutes after ephrinA5-Fc stimulation. Plotted are averaged ratios of the adherent cell surface after and before stimulation. Statistical significance to EphA2 and EphA4 samples (one-way ANOVA and Tukey’s post hoc test) is shown with blue and red stars, respectively. A two-tailed T-test was used to calculate significance between A2A4 and A4A2. Error bars denote s.e.m. * P < 0.05, ** P < 0.01, **** P < 0.0001. c) Confocal time lapse images. Eph clustering was induced by ephrinA5-Fc stimulation in COS7 cells. d) Single molecule experiments were performed using localization microscopy. Normalized Ripley’s L functions (L(r)-r)) are plotted over the distances (r(nm)) between the molecules. Increased (L(r)-r)) at particular distances reflect the amount of clustering and the cluster sizes. e) Further quantification of data presented in panel d. The percentage of detected molecules present in clusters is shown. Clusters were defined as molecules having at least 20 neighbouring molecules within a radius of 50 nm . f) Cell rounding in response to pre-clustered ephrinA5-Fc and cell adhesion to ephrinA5-Fc containing stripes was measured using transfected HeLa cells. EphA2 blue, EphA4 red, EphA2 su grey. Statistical significance was determined using one-way ANOVA and Tukey’s post hoc test. Error bars denote s.e.m. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: EphrinA5-Fc (R&D Cat. no. 374-EA) at 0.2 mg ml −1 in PBS was mixed with goat anti-human IgG Fcγ (Jackson ImmunoResearch) and imaging medium in a 5:1:28 ratio, and incubated for 2 hours at room temperature.

Techniques: Construct, Binding Assay, FLAG-tag, Two Tailed Test, Microscopy, Transfection