Dynamics of RhoA activation in advancing and collapsing growth cones. (a and b) Time series showing RhoA activation in advancing and collapsing growth cones from differentiated N1E-115 cells cotransfected with the RhoA2G FRET probe and pLenti-Lifeact-mCherry. (top) Ratio (RhoA activation) images. (bottom) Lifeact-mCherry (F-actin) images. Time scale, min:s. Bar, 10 µm. (c and d, top left and right) High-magnification images of advancing and collapsing growth cones outlined in the top rows of panels a and b. Red arrowheads point to the RhoA activity zone. (bottom left) Overlay of the time series frames showed in the top rows of panels a and b. Red arrows indicate the advance or collapse direction. (bottom right) Overlay images of the top-left and right panels of c and d highlight the RhoA activity zone (red) over the filopodia F-actin bundles (green). Red arrowheads point to the RhoA activity zone. Bar, 2 µm. (e) High-resolution images of advancing and collapsing growth cones from differentiated N1E-115 cells stained for phalloidin and pMLC are shown. Bar, 10 µm.