Figure 2

Intact actin and microtubule cytoskeleton are both required for netrin-1-induced protein synthesis. Stage 24/25 retinal explants were treated with control medium, cytochalasin D (CytoD), latrunculin B (LB), colchicine (Colc), or nocodazole (Noco) for 5 min, followed by stimulation with either control medium or netrin-1 for a further 5 min. Protein synthesis in growth cones was measured by methionine analog L-azidohomoalanine (AHA) incorporation and visualized with fluorescent microscopy (A-I). Protein synthesis in growth cones treated with control medium (A) was markedly increased 5 min following treatment with netrin-1 (B). However, this increase was abolished in growth cones treated with cytochalasin D (C, D) or colchicine (E, F). Protein synthesis within the growth cone was almost completely abolished by pretreatment with cycloheximide (CHX, G) or anisomycin (H). Quantification of fluorescence intensity reveals that netrin-1-induced protein synthesis is inhibited by disruption of either actin or microtubule dynamics (I). The number of growth cones analyzed in each treatment group can be found in the corresponding bar of the graph in panel I. Similarly, protein synthesis, as measured by the incorporation of ³H-leucine in precipitated proteins, was stimulated by netrin-1, but this effect was inhibited by treatment with either cytoclalasin D, latrunculin B, colchicine, and nocodazole (J). ***P < 0.0001 Mann-Whitney test. Scale bar 10 μm.