Actin nucleation by Arp2/3 complex requires hydrolyzable ATP.
(A) As a control, we tested elongation of 2 μM
MgATP-actin from F-actin seeds in the presence of 100 μM ATP, ADP,
and AMP-PNP. Kinetics of polymerization are independent of solution
nucleotide within the first 100 s of starting the reaction but
diverge soon after this, because of the exchange of nucleotide with
solution. We therefore limited our observations to the first 100 s
when using ATP-actin. (B) The Arp2/3 complex induces
polymerization of MgATP-actin in the presence of ATP or ADP-BeF3 but
not AMP-PNP or ADP. We used the fact that nucleotide in solution
exchanges onto Arp2/3 complex within seconds (Fig. 1E)
but takes much longer to exchange onto actin monomers (38) to load one
type of nucleotide onto the Arp2/3 complex, but keep ATP bound to
actin. We mixed 2 μM MgATP-actin with a solution of 10 nM Arp2/3
complex and 1.2 μM N-WASP WA (3-fold above saturating concentration)
with 100 μM ATP, ADP, ADP + 2 mM BeF3, or AMP-PNP.