Article Figures & Data
Figures
- Figure 1.
Effect of protein synthesis inhibition on Sema3A (S3A)-induced collapse. A–D, The ribosomal inhibitors cycloheximide (CHX, 25 μm, A) and anisomycin (Aniso, 5 μm, B; 40 μm, C), and the mTOR inhibitor rapamycin (Rapa, 100 nm, D) reduce the extent of collapse (mean ± SEM) in the presence of 250 ng/ml Sema3A. Filled bars indicate addition of Sema3A. E, In contrast, 1 μm lysophosphatidic acid (LPA)-induced collapse is not affected by rapamycin. F, G, Protein synthesis dependence is not abolished by isolation of axons from the soma (F), nor is the residual collapse abolished by combining protein synthesis inhibitors (G). H, Levels of growth cone protein synthesis fall to undetectable levels after application of each inhibitor. I, Using a lower concentration of NGF (10 ng/ml) collapse induced by 250 ng/ml Sema3A is also inhibited by anisomycin. J, Combining 500 ng/ml Sema3A and 10 ng/ml NGF reduces the effect of protein synthesis inhibition to a nonsignificant level. K, L, Phase-contrast and fluorescence images of phalloidin-stained collapsed (K) and spread (L) growth cones. Scale bar, 5 μm. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
- Figure 2.
Protein synthesis dependence and adaptation to varying Sema3A concentrations. The dose–response curve of axon growth cone collapse in the presence of 0–1000 ng/ml Sema3A, also in the presence of PBS control (continuous line), anisomycin (large dashes), or rapamycin (short dashes). Overlapping error bars are separated horizontally for greater clarity. With Sema3A concentrations in excess of 625 ng/ml, the addition of anisomycin or rapamycin has no significant effect on growth cone collapse.
- Figure 3.
Assessment of pathways downstream of Sema-3A. A, Addition of a neutralizing anti-neuropilin-1 antibody (α-NPN-1, 50 μg/ml) abolishes growth cone collapse induced by 500 ng/ml Sema-3A (S3A). B, Inhibition of GSK-3β by lithium chloride (LiCl, 10 mm) reduces collapse, and the effect is additive with rapamycin. C, Inhibition of PTEN by bpV(pic) (100 nm) is also additive with rapamycin-induced inhibition of collapse. D, The effect of GSK3-β inhibition is confirmed at two different Sema-3A concentrations (250 ng/ml and 500 ng/ml) using the selective GSK-3β inhibitor SB216763 (200 nm), and is additive with rapamycin. E, A higher concentration of SB216763 (1000 nm) and another selective GSK-3β inhibitor, AR-014418 (0.5 μm and 5 μm), also inhibit Sema-3A-induced growth cone collapse. F, Using a lower NGF concentration (10 ng/ml), collapse induced by both low and high Sema3A concentrations (250 ng/ml, 700 ng/ml) is approximately halved by SB216763 (200 nm) or AR-A014418 (0.5 μm). G, Control experiment confirming growth cone collapse is not induced by inhibitors of PTEN [bp(V)pic, 100 nm] and GSK-3β (lithium chloride, LiCl, 20 mm; SB216763, 200 nm; AR-014418, 0.5 μm), nor by the combination of rapamycin and SB216763, nor by DMSO at the solvent concentration used for the latter reagents. H, Control experiment confirming that the GSK-3β inhibitor SB216763 reduces Sema3A-induced collapse of growth cones of transected axons. I, Phosphorylation of 4E-BP1 (% control fluorescence) increases with application of Sema3A (250 ng/ml). It is further increased by addition of SB216763 (200 nm), and reduced below control levels by addition of rapamycin (100 nm). J, Phosphorylation of GSK-3β (% control fluorescence) falls with Sema-3A (250 ng/ml), and is not significantly altered by addition of rapamycin (100 nm). Error bars are SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
- Figure 4.
Schematic of proposed pathways mediating PS-dependent and PS-independent growth cone collapse in response to high/low Sema3A concentrations.
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