FIGURE 4:

Rac accumulates at apCAM target sites, and its activity is required for evoked growth responses. (A) Growth cones were challenged with needle-restrained apCAM beads, fixed at the end of the latency period, and stained for F-actin (phalloidin) or with antibodies to Rac, Rho, or Cdc42 and fluorescent secondary antibodies. (B) Rac1 enrichment at apCAM target adhesions is enhanced by force restraint. Bag cells were injected with fixable Texas red–dextran before apCAM target assay, then fixed during latency and probed with monoclonal Rac1 antibody and fluorescent secondary antibodies for volume-corrected ratio imaging. Positions of restrained and unrestrained beads are noted. (C) Rac enrichment is enhanced at force-restrained beads. Rac enrichment factor is calculated by dividing the Rac/volume ratio of either a restrained bead or unrestrained bead by the Rac/volume ratio of nonbead P-domain region of interest. Numbers in parentheses denote number of experiments. Error bars are SEM. (D) Top row, DIC images of control-evoked growth assay at beginning (0 min) and end (5 min) of the evoked growth response. Bottom row, DIC images of the same growth cone subjected to restrained apCAM bead in media containing 100 μM NSC23766 Rac inhibitor. Red dashed line outlines the C domain. (E) Pictogram of evoked growth responses of individual growth cones in control and Rac-inhibited conditions, plotted as in Figure 3F, showing control latency period (blue diamonds), latency of growth cone responses in 100 μM NSC23766 (filled orange squares), or length of recordings in which no growth response was observed (empty squares). (F) Responses from E shown normalized to control latencies. (G) Rac activity is required for F-actin cup and localization of Rac and WRC protein. Growth cones were fixed at 0.5× latency and stained for F-actin (phalloidin), Rac, or WRC (WAVE2 antibody) in the absence (top row) or presence of 100 mM NSC23766 (bottom row). Scale bars: 10 μm (A and B); 5 μm (D and G).