SORLA overexpression attenuates ephrinA1-induced EphA4 clustering. (A) WT hippocampal neurons at DIV3, untreated or exposed to control Fc for 30 min or ephrinA1-Fc for the indicated time, were fixed and stained for EphA4 (green), F-actin (red), or tubulin (blue). Note that ephrinA1-Fc treatment for 5 min results in EphA4 clustering without growth cone collapse. Bar, 5 µm. (B–D) Evaluation of EphA4 clustering in WT, SORLA TG, and KO hippocampal neurons. (B) WT, SORLA TG, and KO hippocampal neurons were treated with ephrinA1-Fc for 5 min and stained for EphA4 (green) and tubulin (blue). Boxed regions are magnified in the adjacent panels. Bar, 10 µm. (C) EphA4 clustering within F-actin–rich areas was examined by microscopy in WT, SORLA TG, and SORLA KO hippocampal neurons at DIV3 by staining for EphA4 (green), F-actin (red), and tubulin (blue). Bar, 5 µm. EphA4 clusters ≥200 nm within F-actin–enriched growth cone regions were scored in a minimum of four independent dissections/experiments (D); graph represents mean ± SE, from individual experiments; *, P < 0.05; **, P < 0.003; Student’s t test. (E) Total EphA4 fluorescence intensities from individual growth cones scored in D were plotted; graph represents mean ± SE.