Actin Purification and Polymerization.

Muscle acetone powder was made using flash-frozen chicken muscle from a local Trader Joe’s grocery store (50). Actin was purified using one cycle of polymerization and depolymerization followed by gel filtration through Sephacryl S-300 and stored in Ca-G-buffer (2 mM Tris⋅HCl, pH 8.0; 0.2 mM ATP; 0.1 mM CaCl₂; 1 mM NaN₃; 0.5 mM DTT). Ca-ATP-actin was converted to Mg-ATP actin by incubation in G-Buffer with 50 µM MgCl₂ and 0.2 mM EGTA (14). We made AMPPNP-actin (16) by first removing free ATP with neutralized AG1-X4 resin (Bio-Rad) and then incubating actin in 1 mM AMPPNP pH 7.0 for 3 min at 4 °C in Mg-G-buffer. We made Mg-ADP-actin by incubating Mg-ATP actin monomers with 1 mM glucose and 5 units/mL hexokinase at room temperature for 10 min followed by adding 0.01 volumes of 100 mM ADP (pH 7.0) alone or with 0.04 volumes of 500 mM potassium phosphate (pH 7.0) (14–16). We purchased ATP (A2383), AMPPNP (A2647), ADP (A2754), and hexokinase (H6380) from Sigma-Aldrich, AG 1-X4 resin (1431345) from Bio-Rad, and glucose (167454) from Thermo Fisher Scientific.