ARP2 3 COMPLEX FROM ACANTHAMOEBA FINDS PROFILIN AND CROSS-LINKS ACTIN-FILAMENTS/

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with p rofilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be che mically cross-linked to the actin-binding site of profilin. By analyti cal ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 compl ex with a K-d of 7 mu M, an affinity intermediate between the low affi nity of profilin for barbed ends of actin filaments and its high affin ity for actin monomers. These data suggest the barbed end of Arp2 is e xposed, but Arp2 and Arp3 are not packed together in the complex exact ly like two actin monomers in a filament. Arp2/3 complex also cross-li nks actin filaments into small bundles and isotropic networks, which a re mechanically stiffer than solutions of actin filaments alone. Arp2/ 3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another f ilament cross-linking protein. Based on localization and actin filamen t nucleation and cross-linking activities, we propose a role for Arp2/ 3 in determining the structure of the actin filament network at the le ading edge of motile cells.