Differences in actin disassembly between networks constructed with wild-type vs. ATP hydrolysis mutant Arp2/3 complex become apparent under recycling conditions. (A) The addition of cofilin and profilin to the in vitro actin-based motility system causes turnover and recycling of actin monomers by promoting disassembly of actin filaments. Cofilin severs the actin shell, detaching it from the tail. (B and E) Severing of shells from growing tails occurs 14 min earlier in networks built with wild-type Arp2/3 complex (top) than in networks built with double ATP hydrolysis mutant Arp2/3 complex (bottom). (C) Shells built with ATP hydrolysis mutant Arp2/3 grow to longer lengths than shells built with wild-type Arp2/3 complex. (D) Actin intensities of shells constructed with wild-type Arp2/3 are comparable or slightly lower than intensities of shells constructed with ATP hydrolysis mutant Arp2/3. (E) Quantification of the timing of tail severing for WT (13 tails), Arp2Q137A (9 tails), Arp3Q137A (9 tails), and double ATP hydrolysis mutant (8 tails). Arp2Q137A, Arp3Q137A, and double mutant tails result in shells severed later than wild-type tails by 3.5, 4.5, and 12 min, respectively. Bar, 10 µm.