Cytoskeleton Dynamics

How does Arp2/3-mediate the nucleation of branched filaments?

The Arp2/3 complex is composed of 7 evolutionarily conserved subunits (Arp2, Arp3, ARPC1-C5) that are structurally similar to the barbed end of actin [1]. The complex is inherently inactive, however once activated it facilitates the nucleation of actin monomers from existing filaments as new branches or daughter filaments (reviewed in [2]).

The Arp2/3 complex is essential in the formation of lamellipodia, where it facilitates nucleation exclusively at the leading edge (as opposed to regions further away from the front). Although it is absent from the shafts of filopodia [3], it is known to play a critical role in initiation and growth of these structures [4]. Conversely, the Arp2/3 complex is present along the length of shafts of invadopodia. It has also been detected at the base of invadopodia, whilst being absent from the tips [5].

The Arp2/3 complex binds to the sides and pointed ends of pre-existing actin filaments [5], in a step which contributes to its activation [2]. Binding to *nucleation promotion factors (NPFs) occurs via central/acidic (CA) sequences and is also essential in the activation of the Arp2/3 complex. Generally, cells contain a large number of inactive NPFs. Stimuli such as chemoattractants influence signaling pathways to regulate the local concentration of active NPFs near the cell membrane [6][7][8] (reviewed in [9]). A variety of proteins engaged in these signaling pathways are able to regulate NPF activation, such as the Rho GTPase Cdc42, which regulates the NPFs WAVE and WASp. Alternatively, NPFs can be auto-regulated by intramolecular interactions between the WCA (WASP homology 2-Connector-Acidic) and other upstream domains of the NPF, as is the case with WASp and N-WASp [2].

The exact mechanism of nucleation remains unclear however it is believed that all seven subunits coordinate to anchor the pointed end of the new filament at a 78° angle to the existing actin network [10]. NPFs act to deliver actin subunits to the Arp2/3 complex at the barbed end, via binding through their WH2 domain – this serves to push the leading edge of a cell forward [11]. It has also been suggested that each component of the complex plays specific roles, with Arp2 and Arp3 interacting with the pointed end of the daughter filament and ARPC2 and ARPC4 binding to the original filament [10].

Importantly, the compressive stresses placed upon actin filaments may cause them to bend and curve. This property of the actin cytoskeleton was shown to regulate Arp2/3 mediated nucleation [12]. Here, a significantly higher frequency of filament branches were observed originating from the convex side of curved filaments compared to the concave side. This indicated that nucleation was more likely to occur if an extensional strain existed on the Arp2/3 binding surface on the filament compared to a compressional strain, which generates the concave surface [12].

This bias of Arp2/3 towards branch formation on convex curvature was proposed to represent a mechanosensing mechanism whereby detection of filament curvature alters the local cytoskeleton in order to reinforce it against the imposing forces [12].

What are tandem-monomer-binding nucleators?

Although the most commonly described nucleators are the Arp2/3 complex, and the formins, a third group, known as ‘tandem-monomer-binding nucleators’, has also been identified. Each member possesses tandem repeats of G-actin binding motifs.

Included in this group of nucleators are the Spire proteins, Cordon-bleu (Cobl), Leiomodin (Lmod-2), JMY and adenomatous polyposis coli (APC). Common to each of these proteins are repeats of the actin binding motif Wiskott-Aldrich Syndrome protein (WASp) homology 2 (WH2) domain. However, additional actin binding motifs may be present in the individual members, providing variation not their mechanisms of nucleation, but importantly, in the cellular functions they facilitate.

References

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3. Svitkina TM, and Borisy GG. Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J. Cell Biol. 1999; 145(5):1009-26. [PMID: 10352018]
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9. Stradal TEB, Pusch R, and Kliche S. Molecular regulation of cytoskeletal rearrangements during T cell signalling. Results Probl Cell Differ 2006; 43:219-44. [PMID: 17068974]
10. Rouiller I, Xu X, Amann KJ, Egile C, Nickell S, Nicastro D, Li R, Pollard TD, Volkmann N, and Hanein D. The structural basis of actin filament branching by the Arp2/3 complex. J. Cell Biol. 2008; 180(5):887-95. [PMID: 18316411]
11. Pollard TD, and Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003; 112(4):453-65. [PMID: 12600310]
12. Risca VI, Wang EB, Chaudhuri O, Chia JJ, Geissler PL, and Fletcher DA. Actin filament curvature biases branching direction. Proc. Natl. Acad. Sci. U.S.A. 2012; 109(8):2913-8. [PMID: 22308368]