Gulp1 regulates EphB2/ephrinB1 trogocytosis through dynamin. (A) Representative images showing Dyn2 enriches at ephrinB1 clusters during forward trogocytosis in HeLa cells. Responder cells (green dashed line) overexpressing Flag-EphB2 with WT Dyn2 (GFP-Dyn2) were cocultured with ephrinB1ΔC-mCherry⁺ donor cells (red dashed line). (B) Representative images showing endogenous Dyn2 and GFP-Gulp1 colocalize at ephrinB1 clusters during forward trogocytosis in HeLa cells. Responder cells (green dashed line) overexpressing Flag-EphB2 with GFP-Gulp1 were cocultured with ephrinB1ΔC-mCherry⁺ donor cells (red dashed line), and endogenous Dyn2 was immunostained. In A and B, white arrows indicate internalized vesicles and yellow arrows indicate surface clusters. (B′) Pearson’s correlation coefficient analysis for the colocalization between endogenous Dyn2 and GFP-Gulp1-FL or GFP-Gulp1ΔC, and between Life-Act and GFP-Gulp1ΔC at ephrinB1ΔC clusters for experiments described in B, C, and Fig. S3 D, respectively. Maximum fluorescence intensities of Dyn2, Life-Act, and GFP-Gulp1 or GFP-Gulp1ΔC within each ephrinB1ΔC cluster regions of interest were measured and normalized to their respective cell background signal. n = 3 independent experiments, 4–12 cells per experiment. *, P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test. (C) Representative images showing Gulp1ΔC disrupts endogenous Dyn2 recruitment to ephrinB1 clusters. Responder cells (green dashed line) overexpressing Flag-EphB2 with GFP-Gulp1ΔC were cocultured with ephrinB1ΔC-mCherry⁺ donor cells (red dashed line) and endogenous Dyn2 was immunostained. Enlarged insets show reduced Dyn2 signal at ephrinB1 clusters with Gulp1ΔC colocalization (C′) compared with clusters without Gulp1ΔC colocalization (C″). Arrows indicate ephrinB1ΔC clusters. (D) Quantification of Dyn2 and Gulp1ΔC enrichment at ephrinB1 clusters. Maximal fluorescence intensities of Dyn2 and GFP-Gulp1ΔC within each ephrinB1 cluster regions of interest were measured and normalized to background (value from a region in the same responder cell without ephrinB1 clusters or vesicles). Each point represents an independent cluster. Threshold to indicate enrichment was set to 2.5 times that of background. n = 4 independent experiments, 4–10 cells per experiment. (E) Coimmunoprecipitation and Western blot analysis showing the formation of EphB2/Gulp1/Dyn2 complexes during forward trogocytosis, and Gulp1ΔC blocking recruitment of Dyn2 to EphB2 complexes. Responder HeLa cells expressing Flag-EphB2, in combination with myc (as a control [Con]), full-length myc-Gulp1 (FL), or myc-Gulp1ΔC (ΔC), were treated with either DMSO or Dyngo-4a before being cocultured with ephrinB1ΔC-mCherry⁺ donor cells. (F and G) Representative images (F) and quantification (G) showing inhibition of dynamin activity blocks forward trogocytosis in HeLa cells. Quantification results shown as mean ± SEM (n = 3 independent experiments, 9–20 responder cells per condition per experiment, normalized to median GFP⁺ DMSO value per experiment). *, P < 0.05; **, P < 0.01; one-way ANOVA with Tukey’s multiple comparisons test. Scale bars in A–C and F, 10 µm.