ATP hydrolysis is necessary for timely detachment of Arp2/3 from the lamellipodial actin network, as seen from the ATP hydrolysis mutant’s longer lifetime and longer distance traveled. Arp2/3 containing a single ATP hydrolysis mutant subunit behaves differently than wild-type Arp2/3 within the same cell. (A) We expressed wild-type mCherry-tagged Arp2 (or Arp3) along with ATP hydrolysis mutant GFP-tagged Arp2 (or Arp3) in the same cells. (B and C) Imaging of stable S2 cell lines (1-3): Arp2(WT)-GFP, Arp2(WT)-mCherry (30 cells, 907 GFP speckles, 244 mCherry speckles); and (1-4): Arp2Q137A-GFP, Arp2(WT)-mCherry (34 cells, 972 GFP speckles, 321 mCherry speckles). (D and E) Imaging of stable S2 cell lines (2-6): Arp3(WT)-GFP, Arp3(WT)-mCherry (41 cells, 915 GFP speckles, 692 mCherry speckles.); and (2-7): Arp3Q137A-GFP, Arp3(WT)-mCherry (45 cells, 1,673 GFP speckles, 952 mCherry speckles). We created kymographs showing speckle trajectories, which we used to determine speckle distances traveled and speckle lifetimes. Median values for speckle distances traveled (B and D) or speckle lifetimes (C and E) are noted. Each imaging experiment was repeated on three separate occasions and speckle data were combined. (F) As in Fig. 3, F and G, we replotted our data in a box-and-whisker plot and used the nonparametric Kolmogorov-Smirnov test to compare ATP hydrolysis mutant data against WT data. We calculated p-values to be less than 0.001.