Figure 3

Netrin-1-induced increase in eIF4E-BP1 phosphorylation requires an intact actin but not microtubule cytoskeleton. A brief schematic of signaling from netrin receptor leading to protein synthesis (A). Stage 24/25 retinal explants were cultured for 24 h, then treated with the actin depolymerizing agents cytochalasin D (CytoD) and latrunculin (B) (LatB), or the microtubule depolymerizing agents colchicine (Colc) and nocodazole (Noco) for 5 min, followed by stimulation with either control medium or netrin-1 for a further 5 min. To assess the activation of translation, retinal cultures were stained for phospho-eIF4E-BP1 (p-eIF4E-BP1; growth cones in the top row of panel (B)) and the intensity of the immunofluorescence signal was measured per unit area (quantified in (C)). Compared to the control (top row of (B), far left panel) netrin-1 induced a significant increase in P-eIF4E-BP1 signal intensity (top row of (B), cytoskeleton intact), which was completely blocked upon pretreatment with actin inhibitors (top row of (B), actin-disrupted), but not with microtubule inhibitors (top row of (B), microtubule-disrupted. Quantification of p-eIF4E-BP1 signal intensity is shown in (C). *P < 0.05 Mann-Whitney test. In contrast, levels of total eIF4E-BP1 signal intensity (bottom row of (B)) were not significantly affected by netrin-1 or the pharmacological inhibitors. The number of growth cones analyzed in each treatment group can be found in the corresponding bar of the graphs in panels (C) and (D). Scale bar 10 μm.