Figure 3.

Rac is required for EphB2ΔC trans-endocytosis into primary neurons. (A) Representative time course showing the effects of Rac inhibitor EHT1864 versus vehicle control on EphB2ΔC trans-endocytosis into embryonic day 15.5 mouse cortical neurons. At 1 day in vitro, neurons were stained with CellTracker green, treated for 4 h with either vehicle or EHT1864 at the indicated concentrations, and co-cultured with EphB2ΔC-mCherry⁺ HeLa cells (donor cells, shown in red). Top rows of each condition show donor cell alone, and contact sites and subsequent vesicles are marked by arrows. Time-lapse movies were acquired for 3 h with 3-min intervals between images. Deconvolved image of a single focal plane. Bars: 10 µm; (inset) 5 µm. Elapsed time shown as minutes:seconds. (B) Quantification of A. EphB2 uptake was scored manually from time-lapse images. Results are shown as fraction of contact sites (EphB2 clusters) that resulted in uptake (separated vesicle as seen in last image of top row of A). Data represent mean ± SE (n = 4 independent experiments, 5–12 cells per condition per experiment); *, P < 0.05; **, P < 0.01; ***, P < 0.001, repeated measures one-way ANOVA with Dunnett’s post hoc test.