Figure 4

MLCK and ROCK mediate Slit and Netrin-1-dependent growth cone collapse. (A-D) Cultures of dissociated ventral hindbrain neurons. (A,B) Cultures immunostained with anti-neurofilament antibodies (red) and anti-Islet1/2 antibodies (green; single channel fluorescence image in inset). (C,D) Higher power view of growth cones immunostained using Alexafluor-568-phalloidin to show typical uncollapsed (C) and collapsed (D) morphology. In (C) the growth cone has a clear central domain, filopodia and lamellipodia, whereas in (D) the collapsed growth cone appears as a spike. (E,F) Histograms representing the mean percentage growth cone collapse in cultures treated with or without ligands and the inhibitors ML-7 (MLCK) or Y27632 (ROCK) or Blebbistatin (myosin II) (1 to 10 μM). Error bars indicate estimated standard mean. Scale bar = 20 μm (A), 10 μm (B) or 5 μm (C,D).