The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension.
Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.
Bailly
M
M
Albert Einstein College of Medicine, Department of Anatomy and Structural Biology, 1300 Morris Park Avenue, Bronx, NY 10461, USA. m.bailly@ucl.ac.uk
Ichetovkin
I
I
Grant
W
W
Zebda
N
N
Machesky
L M
LM
Segall
J E
JE
Condeelis
J
J
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011554:Pseudopodia / Q000502:physiology; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D009419:Nerve Tissue Proteins; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal