A–F. Aliquots from each reaction were Latrunculin B/phalloidin arrested 15, 30, 45, and 60 seconds after mixing, fractionated, and analyzed as described. Conditions: 5.8 µm ActA-coated beads mixed with 3 µM actin (5% TMR-labeled), and Arp2/3 and CP as indicated.
A. Representative shells after arrest, fractionation, and washing.
B. Rate of shell growth decreases with [Arp2/3] and increases with [CP]. Error bars represent standard deviation (average n=27).
C. Rate of actin assembly is independent of [Arp2/3] and [CP].
D. Rate of filament nucleation is independent of [Arp2/3] but increases with [CP]. Nucleation rate per square micron of bead surface was derived from the slopes of the time courses.
E. Two alternate mechanisms for increasing the density of a dendritic actin network imply different actin assembly kinetics. See text for details.