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. 1999 Mar 30;96(7):3739-44.
doi: 10.1073/pnas.96.7.3739.

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

L M Machesky  1 , R D Mullins, H N Higgs, D A Kaiser, L Blanchoin, R C May, M E Hall, T D Pollard
Affiliations

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

L M Machesky et al. Proc Natl Acad Sci U S A. .

Abstract

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament-a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

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Figures

Figure 1
Figure 1
Proteins used in this study. (A) Linear diagrams of Scar and WASp domains. (B) SDS/PAGE of 10 μg of purified Arp2/3 complexes. A, amoeba Arp2/3 complex A; B, amoeba Arp2/3 complex B. H, human Arp2/3 complex. The gel is stained with Coomassie blue
Figure 2
Figure 2
Effects of Scar constructs, Arp2/3 complex, and profilin on the time course of actin polymerization. (A) No profilin. (B) Acanthamoeba profilin-II at 7.5 μM. (A and B) Conditions: 2.8 μM Acanthamoeba actin monomers, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.1 mM CaCl2, 0.2 mM ATP, 3 mm NaN3, and 10 mM imidazole (pH 7.0) at 22°C. The polymerization of actin was monitored by pyrene fluorescence. (●) No additions to actin; (○) 100 nM Arp2/3 complex; (▴) 50 nM full-length Scar/100 nM Arp2/3 complex; (▵) 50 nM Scar-PWA/100 nM Arp2/3 complex; (▪) 50 nM Scar-WA/100 nM Arp2/3 complex; (▫) 50 nM Scar-W/100 nM Arp2/3 complex; (▾) 50 nM Scar-A/100 nM Arp2/3 complex; (▿) 50 nM Scar-PWA (A) or 50 nM full length Scar (B). Without Arp2/3 complex, these concentrations of Scar constructs have no effect on actin polymerization in either the presence or absence of profilin-I or profilin-II.
Figure 3
Figure 3
Dependence of the activation of actin nucleation by Arp2/3 complex A on the concentrations of Scar-PWA (A) and Scar-WA (B). Conditions: 3.3 μM actin and 0.9 μM amoeba Arp2/3 complex A with Scar-WA or Scar-PWA as indicated by the micromolar concentrations beside each curve. The buffer included 50 mM KCl, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole (pH 7.5). Insets indicate the time (in seconds) to half-maximal polymerization vs. concentrations of Scar-WA or Scar-PWA.
Figure 4
Figure 4
The effect on the time course of polymerization of preincubating Arp2/3 complex and/or Scar constructs with actin filaments before the addition of actin monomers. The reaction was carried out in three steps as described in Materials and Methods. The concentrations given are those after a 6.7-fold dilution of the preincubated components into pyrenyl-actin monomers. Final conditions: 4 μM amoeba actin monomers, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.1 mM CaCl2, 0.2 mM ATP, and 3 mM NaN3. (A) Tris-Cl (pH 8.0) at 10 mM. (×) Actin monomers alone; (▪) actin monomers with 29 nM Scar-PWA/15 nM amoeba Arp2/3 complex A; (▴) actin monomers with 300 nM actin filaments; (●) actin monomers with 300 nM actin filaments preincubated with 29 nM Scar-PWA; (♦) actin monomers with 300 nM actin filaments preincubated with 15 nM amoeba Arp2/3 complex A; (○) actin monomers with 300 nM actin filaments preincubated with 29 nM Scar-PWA/15 nM amoeba Arp2/3 complex B for 1 min; (▫) actin monomers with 300 nM actin filaments preincubated with 58 nM Scar-PWA/15 nM amoeba Arp2/3 complex A for 3 min; (▵) actin monomers with 300 nM actin filaments preincubated with 58 nM Scar-PWA/15 nM amoeba Arp2/3 complex A for 5 min. (B) Imidazole (pH 7.0) at 10 mM. The symbols used are defined in A, except that ○ indicates actin monomers with 300 nM actin filaments preincubated with 400 nM phalloidin/29 nM Scar-PWA/15 nM amoeba Arp2/3 complex A for 1 min.
Figure 5
Figure 5
Dendritic nucleation hypothesis. The association of Arp2/3 complex with Scar and the side of an actin filament activates nucleation of a new actin filament, which is capped at its pointed end by the Arp2/3 complex and which grows in the barbed direction as a branch of the older filament. The exact order of events is not yet known. Scar could bind to Arp2/3 complex before their association with the side of an actin filament.
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