Fig 2: 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.

Fig 3: Role of Gulp1 in Xenopus gastrulation. (A) Expression of XGulp1 protein in vegetal cells. Anti-Gulp1 antibody staining without or with knockdown by XGulp1-MO (left and middle panels) and localization of XGulp1-GFP (300 pg, the right panel). Scale bar, 50 µm. (B) Mid-sagittal fractures of Xenopus early gastrulae. The central endodermal blastocoel floor is curved in uninjected, XGulp1-GFP–injected, or XGulp1-MO/XGulp1-GFP–coinjected embryos, but straight in Gulp1ΔC expressing or XGulp1-MO gastrulae (dashed lines; XGulp1-MO, 30 ng; XGulp1-GFP, 900 pg). Red arrowheads, dorsal blastopore; BCR, blastocoel roof; LEM, leading edge mesendoderm that begins to advance on the BCR; V, endoderm of vegetal cell mass. Scale bar, 500 µm. (C) Curvature of blastocoel floor. Embryos from three independent experiments were measured by the Kappa plugin in ImageJ, and results were pooled for each treatment. (D) Explants of mid-sagittal slices of vegetal cell mass fixed immediately after excision (0 hr) and after onset of vegetal rotation (1 hr) uninjected (top) or after Gulp1ΔC mRNA injection (bottom). Yellow dashed lines, endodermal blastocoel floor surface. Average lengths of dashed lines from three independent experiments: 2,381.0 ± 254.5 µm for eight uninjected explants; 1,862.0 ± 285.5 µm for nine Gulp1ΔC mRNA injected explants. Red arrowheads, dorsal blastopore; green arrowheads, border between surface of embryo and blastocoel floor. Scale bar, 200 µm. (E) Cells in vegetal slice explants after injection of ephrinB1-mCherry mRNA alone or together with XGulp1-MO or XGulp1-MO and XGulp1-GFP. Amount of mRNA injected per blastomere is indicated. Explants from three sets of experiments were examined by confocal microscopy. Additional explants analyzed by conventional fluorescence microscopy gave the same results (not shown). White arrowheads point to retracting protrusions, yellow arrowheads to cell regions simultaneously enriched in vesicles and XGulp1-GFP. Scale bars, 50 µm (left) and 30 µm (right). (F and G) Number of intracellular dots per cell. Asterisks indicate significance levels: ****, P < 0.0001; not significant, P = 0.286; two-tailed Student’s t test.

Fig 4: Gulp1 interacts with EphB2. (A) Validation of the interaction between Gulp1 and EphB2 using coimmunoprecipitation. HeLa cells overexpressing GFP-Gulp1 and either full-length Flag-EphB2 (Flag-EphB2-FL) or Flag-EphB2ΔC were either not treated (control) or treated with either preclustered Fc or preclustered ephrinB1-Fc, or cocultured with HeLa cells overexpressing ephrinB1ΔC-CFP (ephrinB1ΔC). (B) Model of EphB/ephrinB forward trogocytosis. EphrinBΔC from donor cells is trans-endocytosed into EphB+ responder cells. (C) Representative images showing forward trogocytosis in U251 cells (magenta dashed line, labeled with Cell-tracker) cocultured with ephrinB1ΔC-GFP+ donor HeLa cells (right panels, green dashed line), but not control GFP+ cells (left panels, green dashed line). Internalized ephrinB1ΔC vesicles in U251 cells were detected as green puncta void of surface HA-antibody labeling (arrows). Scale bars, 10 µm. (D) Validation of the interaction between endogenous Gulp1 and EphB2. U251 cells were first cocultured with either control GFP+ or ephrinB1ΔC-GFP+ HeLa cells for 30 min, and cell lysates were then subjected to immunoprecipitation by anti-EphB2 antibodies. (E) Representative images from live imaging of forward trogocytosis in HeLa cells. EphrinB1ΔC-mCherry+ donor cells were cocultured with responder cells expressing untagged EphB2 and GFP-Gulp1. Middle row: green dashed lines indicate the responder cell outline. Bottom rows show time course at time of contact and scission. Arrows indicate ephrinB1ΔC cluster formation and subsequent vesicle internalization. GFP-Gulp1 images pseudo-colored as heat maps (bottom row). Scale bars, 10 µm (top panel), 5 µm (inset), and 2 µm (time-lapse images). Elapsed time shown as min:s. (F) Average fluorescent intensities at contact sites of cluster formation (F′) and subsequent vesicles (F″) from time-lapse imaging of cocultures as described in E. Data displayed as heat maps of average intensity calculated over every event, with donor contact sites for each set aligned to top and center, and all images normalized to their respective background signal. Graphs show mean ± SEM of relative fluorescent units (RFU) changes through the central four pixels for the x axis. Data acquired from 32 events from 10 cells over two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; one-way ANOVA with Bonferroni’s multiple comparisons test performed on GFP-Gulp1 signal.

Fig 5: Gulp1 regulates the EphB2/ephrinB1-mediated cell disengagement response. (A–C) Time-lapse images (A and B) and quantification (C) showing expression of dominant-negative Gulp1ΔC inhibits EphB2/ephrinB1-mediated cell disengagement. HeLa cells expressing EphB2 and either GFP or GFP-Gulp1ΔC were cocultured with donor cells expressing ephrinB1ΔC-mCherry. Maximum projection of deconvolved images is shown. Scale bars, 10 µm. Elapsed time shown as min:s. Dashed lines indicate the distance between two contacting cells. Measured distance between donor and responder cells over time for each condition shown as mean ± SEM n = 20 (GFP-Gulp1ΔC) and 13 (GFP) donor–responder pairs from three experiments. ***, P < 0.001; two-way ANOVA.