Fig. 6. PTEN accumulates at the growth cone membrane during Sema3A mediated growth cone collapse. (A) DRG neurons from E18 rat embryos were nucleofected with GFP-PTEN and cultured overnight in vitro. Growth cones were imaged by confocal time-lapse microscopy at 1 frame every 30 seconds. The presented selection of images shows a growth cone 3 minutes before (-3', -2', -1') and during the response to Sema3A, which was applied at 0'. Following Sema3A application, fluorescent signals of GFP-PTEN increase uniformly at the membrane. See also Movie 2 in supplementary material. (B) A 3-pixel section in each frame was taken at the level indicated at 16' in A and pasted side by side. To facilitate the visualisation of movement and changes in relative fluorescence intensity at the membrane over time, we applied pseudo colour (see arrowhead). Following Sema3A addition at 0' (arrow) relative levels of PTEN increased at the membrane after 4 minutes (asterisk). Bar, 10 µm.