FIGURE 1:

Nonbundled-filament barbed-end capture is not processive. Conditions as follows: 1.5 μM (33–40% labeled) Mg-ATP actin, nanofibers coated with 10–20 μM GST-PP-WWCA, 1 mM MgCl₂, 10 mM imidazole, pH 7, 50 mM KCl, 1 mM EGTA, 100 mM DTT, 0.2 mM ATP, 0.25% methyl cellulose, 15 mM glucose, 20 μg/ml catalase, and 100 μg/ml glucose oxidase. Slides coated with NEM-inactivated myosin II. (A) The NWASP constructs used in the study. (B) Elongating barbed ends (arrows) attached to a GST-PP-WWCA–coated, fluorescein isothiocyanate–labeled BSA blocked nanofiber (dashed outline), polymerizing either perpendicularly (white arrow) or in parallel (black arrow). (C) Growth of barbed ends parallel to the nanofiber before and after capture. (D) Growth of barbed ends attached perpendicular to the nanofiber before and after capture. (E–H) Histogram of smoothed growth rates before (E) and after (F) parallel capture of barbed ends or before (G) and after (H) perpendicular capture. (I) Box-and-whisker plot of growth rates of parallel and perpendicular captured filaments. Scale bar, 10 μm.