Dimerizer-induced EphB2 clusters cause GC collapse in neurons. (A) Rat hippocampal cultures were transfected with YFP, ΔNEphB2-0FKBP, or ΔNEphB2-3FKBP, and treated at 1 d in vitro with control stimuli (Fc fragment plus ethanol), 1 µg/ml preclustered ephrinB2-Fc, 250 nM AP20187, or 250 nM AP1887 for 30 min. Neurons were fixed and stained with anti-GFP antibody (green) and phalloidin (red) to visualize f-actin–rich GCs. Insets show GCs indicated by dashed boxes. Only the status of GCs of the longest neurites positive for Tau1 (axons) was quantified. Bar: (main images) 30 µm; (insets) 10 µm. (B) Quantification of GC collapse (mean ± SEM in percentage of all GCs counted). EphrinB2-Fc induced GC collapse in all cultures. AP20187 was as efficient as ephrinB2-Fc in inducing GC collapse in neurons expressing the 3FKBP isoform, whereas stimulation with AP1887 led to an intermediate response (n = 3 independent experiments; ***, P < 0.001; **, P < 0.01; *, P < 0.05; Student’s t test; asterisks in red represent significance level to Fc/EtOH control stimulation of each dataset).