Figure 4.

Time-dependent changes in the behavior of active Rho GTPases. A. Left panels show kymographs (made as in figure 3) of turnover of active Cdc42 at wound edge assessed with PA-GFP-wGBD (green) and fluorescent actin (red) at increasing times after wounding (Time in min:sec indicated on left; each panel from same wound). Right panels show active Cdc42 alone; asterisks indicate persistence peaks. B. Turnover of active Rho at wound edge assessed with PA-GFP-rGBD (green) and fluorescent actin. Same layout and labeling as in 4A. C. Quantification of total (ie entire photoactivated regions) half life of soluble proteins (PA-GFP), active Cdc42 or active Rho. PA-GFP was assessed in unwounded cells (no wound), in wounded cells but at > 20 μm away from the wound (off wound) or at the wound edge after the onset of contraction (Wound Edge). Active Cdc42 and Rho were assessed in wounded cells at > 20 μm away from the wound (Off wound), at the wound (ie within their zones) before the onset of contraction (Precontraction) or at the wound after the onset of contraction (Contraction). Asterisks indicate P < 0.05; numbers indicate N. Bars indicate S.D. D. Comparison of the front:back total half life ratio for active Cdc42 and Rho before (Before flow) and after (After flow) the onset of contraction. E. Comparison of the contractile ring closure velocity when the front-to-back half-life ratio is less than 1 or greater than 2 for either active Cdc42 or active Rho. F. Kymographs (made as in Fig. 3) showing examples of active Cdc42 and active Rho undergoing forward translocation. Dotted yellow line shows the leading edge of the photoactivated probe; blue dotted line shows expected position of photoactivated probe if it remains stationary. G. Comparison of velocity of forward movement of forward translocating active Cdc42 and active Rho versus velocity of contractile ring closure in the same sample.