Fig. 1

Actin, but not microtubule, disruption blocks netrin-1 induced PI3K/Akt/mTOR signaling in growth cones. Cultured stage 24/25 retinal explants were treated with a DMSO vehicle control, cytochalasin D (CytD), or colchicine (Colc) for 5 min, followed by 5-min stimulation with either control medium or netrin-1. Quantitative immunofluorescence showed that levels of activated mTOR (p-mTOR; (a)) and Akt (p-Akt; (e)) were elevated by netrin-1 stimulation, a process that was prevented by actin, but not microtubule disruption (quantified in (c) and (g)). Total levels of mTOR (b) and Akt (f) were not significantly altered following stimulation with netrin-1, in either untreated or growth cones treated with cytoskeletal disrupting agents (quantified in (d) and (h)), with the exception of total Akt levels in growth cones treated with colchicine, which showed a small, but significant increase in fluorescence intensity. The number of growth cones analyzed in each treatment group can be found in the corresponding bar of the respective graphs. **P < 0.005; ***P < 0.0001 Mann–Whitney test. (i) Pseudocolored images of a live PI3K biosensor (PHAkt-GFP) before and after netrin-1 stimulation in the presence of cytoskeletal inhibitors. Growth cones expressing low levels of PHAkt-GFP were treated with cytoskeletal inhibitors (0.1 μM cytochalasin D or 12.5 μM colchicine) on stage during time-lapse acquisition on an inverted spinning disk confocal system (60× water immersion). Time-lapse imaging showed an increase in PHAkt-GFP signal after netrin-1 stimulation, which was inhibited by cytochalasin D treatment, but not affected by colchicine treatment (j). Background-subtracted fluorescent signals were normalized to the control medium at time 0. Scale bar 10 μm