Abstract
Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.
Highlights
The Arp2/3 complex is a key cytoskeletal regulator of actin polymerization [1]
The isolated complex has a low nucleation activity, but upon binding to nucleation promoting factors (NPFs), ATP, and preexisting actin filaments, the Arp2/3 complex promotes the formation of a branched actin structure where the complex itself is situated at the branch junction [3,4]
We show that various NPFs, except cortactin, dissociate from the complex after branch formation and that all of the Arp2/3 subunits are in a position to contact the mother filament
Summary
The Arp2/3 complex is a key cytoskeletal regulator of actin polymerization [1]. The complex promotes the assembly of dendritic actin networks that drive cell locomotion, phagocytosis, and intracellular motility of lipid vesicles, organelles, and invasive pathogens [2]. Consistent with a conformational change upon activation, the relative orientation of both Arp and Arp would need to be altered in our model to provide an exact match of the daughter filament with the direction of its projection density This conformation could be achieved by an approximately 158 rotation of Arp around its short axis and an approximately 158 rotation of Arp around an axis parallel to its short axis passing through domain I of Arp, accompanied by a slight adjustment of the overall complex orientation (,58). The data from subunit labeling, in conjunction with the crystal structure of the isolated complex, allowed a much more detailed and accurate assignment of the densities than previously possible and indicate that the previous assignment was one unit off (i.e., the previous Arp position corresponds to Arp and Arp to the first actin monomer in the daughter filament)
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