The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments.
The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 microM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 +/- 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.
Mullins
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RD
The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Heuser
J A
JA
Pollard
T D
TD
D000048:Acanthamoeba / Q000378:metabolism* / Q000648:ultrastructure; D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D051377:Actin-Related Protein 2; D000199:Actins / Q000378:metabolism*; D000818:Animals; D003598:Cytoskeletal Proteins; D019281:Dimerization; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism*
D051377:Actin-Related Protein 2; D000199:Actins; D003598:Cytoskeletal Proteins; D015800:Protozoan Proteins