Figure 4.

Gulp1 cooperates with Tiam2 to facilitate EphB2/ephrinB1 trogocytosis. (A and B) Representative images (A) and quantification (B) of coculture assays shows constitutively active Tiam2 (GFP-Tiam2ΔN) boosts the forward trogocytosis gain of function effect seen upon overexpression of Gulp1-FL. Responder cells (green dashed line) overexpressing Flag-EphB2 and myc-Gulp1 or myc (as a control), together with either GFP-Tiam2ΔN or GFP (as control), were cocultured with ephrinB1ΔC-mCherry⁺ donor cells (red dashed line). Scale bars, 10 µm. Relative values of vesicle numbers per cell shown as mean ± SEM (n = 3 independent experiments, 16–34 responder cells per condition per experiment). Data normalized to median GFP/myc value per experiment. ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test. (C) Coimmunoprecipitation and Western blots analysis showing GFP-Tiam2ΔN (ΔN) enhances the interaction between Gulp1 and EphB2. HeLa cells were transfected with Flag-EphB2, in combination with either full-length myc-Gulp1 or myc-Gulp1ΔC, and either GFP-Tiam2ΔN or GFP. (D and E) Representative images (D) and quantification (E) of coculture assays showing GFP-Tiam2ΔN enhances Gulp1ΔC enrichment at ephrinB1ΔC clusters. Responder cells (green dashed lines) overexpressing Flag-EphB2 and myc-Gulp1ΔC, together with either GFP-Tiam2ΔN or GFP (as control), were cocultured with ephrinB1ΔC-mCherry⁺ donor cells (red dashed lines). Arrows indicate ephrinB1ΔC clusters. Percentage of cells with myc-Gulp1ΔC enrichment at ephrinB1ΔC clusters shown as mean ± SEM (n = 3 independent experiments, 17–32 responder cells per condition per experiment). **, P = 0.0099, two-tailed unpaired t test. (F) Quantification of coculture assays showing GFP-Tiam2ΔN potentiates inhibition of forward trogocytosis by Gulp1ΔC. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Tukey’s multiple comparisons test.