Fig. 1.
Independent control of F-actin length and turnover at steady state in vitro. (A) Schematic of the actin-turnover cycle. B and P denote filament barbed and pointed ends, respectively. (B) Schematic of important biochemical activities of actin, profilin, cofilin, and formin. (C) Time course of Pi production for 1.5 µM Mg–ATP–actin alone (gray), with 4.5 µM profilin (Rp = 3; blue), with 0.75 µM cofilin (Rc = 0.5; yellow), or with 4.5 µM profilin and 0.75 µM cofilin (orange), all in the presence of 0.2 mM 2-amino-6-mercapto-7-methylpurine ribonucleoside and 1 U/mL purine nucleoside phosphorylase. (C, Inset) Extended vertical axis showing the linear increase in all samples at long times. (D) Bulk Pi-release rate from linear fits to the Pi-release time course 3–4 h after polymerization is initiated, as indicated by portion shaded in light blue (C, Inset). Error bars indicate SEM; n = 4 for the profilin + cofilin condition, and n = 2 for each of the others. (E) Typical time courses of seeded assembly reactions in which 0.25 µM Mg–ATP–actin monomers (10% pyrene-labeled) are added to 0.5-µM unlabeled actin seeds formed in the absence of additional proteins (gray), with 1.5 µM profilin (Rp = 3; blue), with 0.25 µM cofilin (Rc = 0.5; yellow), or with 1.5 µM profilin and 0.25 µM cofilin (orange). (F) Steady-state filament concentrations calculated using linear fits to the seeded assembly time courses during the time window shaded in light blue in E. Error bars indicate SEM; n = 6, 6, 5, and 6 per condition, from left to right. (G) Single-filament turnover rates calculated from the data in D and F and rescaled by actin concentration for actin alone, with Rp = 3, with Rc = 0.5, or with Rp = 3 and Rc = 0.5. Data are normalized (norm.) by the single-filament turnover rate for actin alone (gray). Error bars denote propagation of error from the mean filament concentration and mean bulk phosphate release-rate measurement for each condition. (H) Length distribution of Alexa 488–phalloidin-labeled filaments from source solutions containing 11.9 µM actin, Rp = 3, Rf = 0.01, and either no cofilin (Rc = 0; blue) or 6.95 µM cofilin (Rc = 0.5; orange). Each length distribution is composed of 100 filaments from each of two independent samples, for a total of 200 filaments per condition. a.u., arbitrary units.