|Eph forward signaling prevents nuclear ERK translocation.|
Wild-type hippocampal neurons were treated with guidance cues as indicated, followed by staining for P-ERK (green), DAPI (blue) and F-actin (red). Arrows point at the nucleus. (A-D) In untreated neurons, nuclear P-ERK levels were low. (E–H) BDNF enhanced P-ERK levels, which accumulated in and around the nucleus. (I–L) Ephrin-A5 did not increase nuclear P-ERK accumulation. However, P-ERK positive clusters were observed in the neurite (arrowhead in I). (M-P) Ephrin-A5, when applied along with BDNF, antagonized nuclear P-ERK entry evoked by BDNF. As with ephrin-A5 (I), P-ERK localized to neurites (arrowheads in M). (Q) Quantification of nuclear P-ERK. The ratio of P-ERK in the nucleus vs. cytosol was plotted against the three time-points of stimulation. Values of ratios for untreated neurons were set to 1. Statistical significance is provided relative to control (untreated) values. (R-U) Total ERK levels remained constant throughout the various treatments. (V) Hippocampal neurons stained for P-ERK localization. Ephrin-A5 stimulation induces clusters of P-ERK in neurites. (W) All Eph receptors bound by ephrin-A5 were labeled. Typically, Eph receptors were found in clusters localized to the neurites. (X) A merged image of (V) and (W), revealing co-localization of P-ERK and Eph receptors in hippocampal neurites. Scale-bar (A-P, R-X) = 10 µm.