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. 2019 Aug 13;218(10):3455–3471. doi: 10.1083/jcb.201901032

Figure 3.

Figure 3.

Gulp1ΔC serves as a dominant-negative protein blocking EphB2/ephrinB1 trogocytosis. (A and A′) Representative confocal image (A) and STED image (A′) of the same cell showing GFP-Gulp1ΔC specifically colocalizes with EphB2/ephrinB1 clusters (visualized by ephrinB1ΔC-mCherry signal, x-y resolution = 20 nm). Scale bars, 20 µm (A), 5 µm (A′, left panel), and 2 µm (A′, right panels). (B and C) Representative images (B) and quantification (C) showing Gulp1ΔC blocks forward trogocytosis in HeLa cells when enriched at EphB2/ephrinB1 clusters. Internalized vesicles detected by comparing total ephrinB1ΔC signal to surface signal. Arrows indicate GFP-Gulp1ΔC enrichment at ephrinB1ΔC clusters; asterisks indicate higher GFP-Gulp1ΔC signal in nucleus. Scale bars, 10 µm. Relative value of vesicles number per cell shown as mean ± SEM (n = 4 independent experiments, 25–40 responder cells per condition per experiment). Data normalized to median GFP value per experiment. The right two columns show the GFP-Gulp1ΔC condition separated into cells that either show GFP-Gulp1ΔC and ephrinB1ΔC colocalization (coloc), or not (no coloc). *, P < 0.05; ***, P < 0.001; ns, not significant; one-way ANOVA with Dunnett’s post hoc test. (D and E) Representative time-lapse images (D) and quantification (E) showing Gulp1ΔC blocks reverse trogocytosis in cultured cortical neurons. Scale bars, 10 µm. Elapsed time shown as min:s. Arrows indicate EphB2ΔC clusters or subsequent trogocytosed vesicles. Relative percentage of contacted neurons (indicated by EphB2 clusters) with internalized EphB2ΔC vesicles shown as mean ± SEM (n = 3 independent experiments, 4–13 neurons per condition per experiment). *, P = 0.0305, two-tailed unpaired t test. (F) Representative images showing Gulp1 is required for reverse trogocytosis in cultured cortical neurons. Neurons were labeled with βIII-tubulin antibodies. Internalized EphB2ΔC vesicles, indicated by white arrows (red puncta), were differentiated from surface EphB2ΔC clusters, indicated by yellow arrows (yellow puncta). Scale bars, 10 µm. (G) Western blot analysis showing efficient knockdown of endogenous Gulp1 expression in primary cultured neurons. (H) Quantification showing percentage of contacted neurons (indicated by EphB2 clusters) with internalized EphB2ΔC vesicles. **, P = 0.0015; two-tailed unpaired t test. (I) Quantification showing relative value of vesicles number per contacted neuron. Data normalized to median control value per experiment. Results shown as mean ± SEM (n = 3 independent experiments, 22–29 responder cells per condition per experiment). ***, P = 0.0009; two-tailed unpaired t test.