Activation of Arp2/3 Complex: Addition of the First Subunit of the New Filament by a WASP Protein Triggers Rapid ATP Hydrolysis on Arp2
Figure 2
ATP Hydrolysis by Arp2 Coincides with Nucleation of New Actin Filaments, and Not Filament Debranching
(A, B) The kinetics of nucleation were slowed by using only 1 μM monomeric actin (compared to 2 μM for Figure 1). γ-32P-AzidoATP-Arp2/3 (20 nM) was mixed with either 750 nM N-WASP WWA (closed circle) or Scar1 WA (closed square) and 1 μM 7% pyrene-labeled monomeric actin.
(A) Actin polymerization measured by pyrene fluorescence.
(B) The concentration of new filament ends (open symbols) was calculated from the polymerization data in a model-independent way (see Methods and Materials), and Arp2-ATP hydrolysis (closed symbols) was measured under the same reaction conditions for both N-WASP WWA (open and closed circles) and Scar1 WA (open and closed squares).
(C) ATP hydrolysis on Arp2 does not accompany filament debranching. Using a large excess (100 nM) of γ-32P-AzidoATP-Arp2/3 creates a slow hydrolysis phase that follows the rapid nucleation phase. The slow phase of ATP hydrolysis can be inhibited by excess (1.5 μM) uncrosslinked Arp2/3 added at t = 200 s, showing that the slow phase of ATP hydrolysis is from Arp2/3 being recruited from solution and not from that already incorporated in branches.