Fig. 4. LatB treatment paradoxically increases free G-actin concentration in the cell. (A) Effects of LatB on actin polymerization mediated by mDia1F2. Representative results showing no obvious promoting effect of LatB on mDia1F2-induced actin assembly. Actin (1 µM, left panel; 3.3 µM, right panel; 20% pyrene labeled) was polymerized in the presence of various concentrations of LatB with 21.8 nM (left) or 10 nM (right) mDia1F2. (B) Simulation analysis reveals a paradoxical increase in free G-actin upon low-dose LatB treatment. This kinetic model (bottom) concerns changes in the concentration of G-actin (G), either free or bound to thymosin-β4 (T), profilin (P) and LatB (L). The graphs show simulation at 0.1 µM LatB, 0.5 µM free G-actin at t=0, 115.5 µM total G-actin at t=0, 240 µM total actin, 140 µM total thymosin-β4 (40 µM free Tb4 at t=0) and 18 µM total profilin (3 µM free profilin at t=0). On the right, curves for five parameters are enlarged. Note the rapid increase in free G-actin despite the accumulation of LatB to 5 µM in the cytoplasm. (C) Estimation of the equilibrium state also predicts an increase in free G-actin. Relative F-actin contents were quantified by phalloidin binding in cells treated with 100 nM LatB for 0, 1 and 2 minutes (line, mean ± s.d. of three separate experiments; n=275 cells, n=245 cells and n=201 cells, respectively). Dashed line and columns show changes in total G-actin and free G-actin, respectively, estimated by calculating the equilibrium state between G-actin and its interacting molecules based on the observed F-actin decrease.