Fig. 4.
BeFx suppresses pointed-end dynamics. (A) Raw kymographs (Upper), and a skeletonized version of each (Lower), illustrating growth of the barbed end (magenta) and the absence of pointed-end dynamics, similar to canonical actins (Movie S6). (B) Time course of growth of 8 PfAct1 filaments in the presence of BeFx shows growth followed by a steady-state phase. (C) Polymerization rate as a function of actin concentration in the presence of BeFx (blue inverted triangles). BeFx slowed both the assembly and disassembly rates, thus maintaining a critical concentration similar to that observed in its absence (red dashed line). The assembly rate constant (slope) was 2.2 subunits/µM·s, the disassembly rate constant (y-intercept) 8.6 subunits/s, and the critical concentration (x-intercept) 4.0 µM. See Table 1 for average polymerization parameters from multiple experiments. (D) Image showing that in the presence of BeFx a large percentage of PfAct1 filaments have a bright, short oligomer at the pointed end (yellow arrows). A number of short oligomers are also seen on the surface. Polymerization buffer: 10 mM imidazole, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 2.5 mM MgATP,10 mM DTT, 0.25% methylcellulose, 0.13 mg/mL glucose oxidase, 50 μg/mL catalase, and 3 mg/mL glucose.