Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP

Luke A Helgeson,Brad J Nolen

doi:10.7554/elife.00884

Abstract

Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation. DOI:http://dx.doi.org/10.7554/eLife.00884.001.

Highlights

Orchestration of many complex cellular processes, including cellular motility, endocytosis, and cytokinesis, requires tight control of the assembly and disassembly of actin filament networks (Chhabra and Higgs, 2007; Pollard and Cooper, 2009)
Previous experiments showed that cortactin activates Arp2/3 complex weakly on its own, but potently synergizes with WASP/N-WASP (Weed et al, 2000; Weaver et al, 2001; Uruno et al, 2001)
We added a range of concentrations of cortactin to a reaction with GST-N-WASP-VCA (GST-VCA) and Arp2/3 complex (Figure 2A)

Summary

Introduction

Orchestration of many complex cellular processes, including cellular motility, endocytosis, and cytokinesis, requires tight control of the assembly and disassembly of actin filament networks (Chhabra and Higgs, 2007; Pollard and Cooper, 2009). Actin-related protein (Arp)-2/3 complex is an important actin cytoskeletal regulator that mediates the assembly of branched actin filament networks by nucleating new (daughter) filaments from the sides of pre-existing (mother) filaments (Figure 1A) (Goley and Welch, 2006; Rotty et al, 2013). When isolated from most species, the complex is inactive, and activation requires binding to the side of a preformed actin filament and association with a nucleation promoting factor (NPF) protein (Figure 1A) (Pollard, 2007; Achard et al, 2010). The mechanism by which multiple NPFs coordinately regulate Arp2/3 complex activity is poorly understood

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