Figure 4.
Figure 4. Degree of EphB2 clustering determines strength of cellular response. (A) Images of representative HeLa cells in bright-field (BF) and fluorescence, expressing equal and moderate levels of wtEphB2 carrying 0 to 3FKBP domains, before (start) and after stimulation with 250 nM AP20187 for 17 min. Cell collapse was scored by measuring the cell surface area (red outlines; see Materials and methods). (top right) Graph showing changes in mean cell area (± SEM from n = 10 cells for each isoform) over time (in percentage relative to the start of the experiment) induced by AP20187. Statistical significance was determined using two-way ANOVA with post hoc Bonferroni test and is shown with black stars (0 versus 2 or 3FKBP) and blue stars (2 versus 3FKBP); ***, P < 0.001; ****, P < 0.0001. (bottom right) Bar graph showing changes in collapse amplitude (minimal surface area in percentage relative to start of experiment) of cells within 40 min after stimulation (mean cell area ± SEM from n = 30 cells for each isoform; **, P < 0.01; ***, P < 0.001, Student’s t test). Bars, 10 µm. (B) Representative Western blot of anti-Flag immunoprecipitated EphB2-3FKBP isoforms using anti-phospho-EphB2 antibodies; blots were stripped and reblotted for total EphB2 levels. Blots show the time course of EphB2 autophosphorylation in transfected HeLa cells induced by the low-affinity dimerizer AP1887, compared with the indicated concentrations of high-affinity dimerizer AP20187, or preclustered ephrinB2-Fc. Note that the kinetics of EphB2 autophosphorylation was slower for AP1887 compared with AP20187. After 20 min, 250 nM AP1887 was as effective as AP20187 and ephrinB2-Fc. In total the experiment was repeated three times with the same outcome. (C) Quantification of collapse responses of cells expressing the wtEphB2-3FKBP isoform induced by the low affinity dimerizer AP1887 compared with AP20187 (each 250 mM; mean cell area ± SEM from n = 17, 16, and 12 cells for conditions AP20187, AP1887, and control). Statistical significance was determined as in A and is shown with black stars (control versus dimerizer) and blue stars (AP1887 versus AP20187). Note that the response induced by AP1887 is slower and weaker. (D) Quantification of collapse responses of cells expressing wtEphB2 induced by equal concentrations (2 µg/ml) of preclustered ephrinB2-Fc and ephrinB3-Fc compared with the low affinity ligand ephrinA5-Fc. As a control, cell collapse of untransfected cells (UNT) was measured upon ephrinB2-Fc stimulation (mean cell area ± SEM from n = 13, 14, 15, and 8 cells for conditions ephrinB2-Fc, ephrinB3-Fc, ephrinA5-Fc, and UNT/ephrinB2-Fc) Statistical significance was determined as in A and is shown with black stars (untransfected versus wtEphB2) and blue stars (ephrinA5-Fc versus ephrinB2-Fc or ephrinB3-Fc).

Degree of EphB2 clustering determines strength of cellular response. (A) Images of representative HeLa cells in bright-field (BF) and fluorescence, expressing equal and moderate levels of wtEphB2 carrying 0 to 3FKBP domains, before (start) and after stimulation with 250 nM AP20187 for 17 min. Cell collapse was scored by measuring the cell surface area (red outlines; see Materials and methods). (top right) Graph showing changes in mean cell area (± SEM from n = 10 cells for each isoform) over time (in percentage relative to the start of the experiment) induced by AP20187. Statistical significance was determined using two-way ANOVA with post hoc Bonferroni test and is shown with black stars (0 versus 2 or 3FKBP) and blue stars (2 versus 3FKBP); ***, P < 0.001; ****, P < 0.0001. (bottom right) Bar graph showing changes in collapse amplitude (minimal surface area in percentage relative to start of experiment) of cells within 40 min after stimulation (mean cell area ± SEM from n = 30 cells for each isoform; **, P < 0.01; ***, P < 0.001, Student’s t test). Bars, 10 µm. (B) Representative Western blot of anti-Flag immunoprecipitated EphB2-3FKBP isoforms using anti-phospho-EphB2 antibodies; blots were stripped and reblotted for total EphB2 levels. Blots show the time course of EphB2 autophosphorylation in transfected HeLa cells induced by the low-affinity dimerizer AP1887, compared with the indicated concentrations of high-affinity dimerizer AP20187, or preclustered ephrinB2-Fc. Note that the kinetics of EphB2 autophosphorylation was slower for AP1887 compared with AP20187. After 20 min, 250 nM AP1887 was as effective as AP20187 and ephrinB2-Fc. In total the experiment was repeated three times with the same outcome. (C) Quantification of collapse responses of cells expressing the wtEphB2-3FKBP isoform induced by the low affinity dimerizer AP1887 compared with AP20187 (each 250 mM; mean cell area ± SEM from n = 17, 16, and 12 cells for conditions AP20187, AP1887, and control). Statistical significance was determined as in A and is shown with black stars (control versus dimerizer) and blue stars (AP1887 versus AP20187). Note that the response induced by AP1887 is slower and weaker. (D) Quantification of collapse responses of cells expressing wtEphB2 induced by equal concentrations (2 µg/ml) of preclustered ephrinB2-Fc and ephrinB3-Fc compared with the low affinity ligand ephrinA5-Fc. As a control, cell collapse of untransfected cells (UNT) was measured upon ephrinB2-Fc stimulation (mean cell area ± SEM from n = 13, 14, 15, and 8 cells for conditions ephrinB2-Fc, ephrinB3-Fc, ephrinA5-Fc, and UNT/ephrinB2-Fc) Statistical significance was determined as in A and is shown with black stars (untransfected versus wtEphB2) and blue stars (ephrinA5-Fc versus ephrinB2-Fc or ephrinB3-Fc).

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