The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments

Abstract

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 microM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 +/- 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

Figure 1

Arp2/3 complex caps the pointed ends of actin filaments with nanomolar affinity. (A) Polymerization of 2 μM 7% pyrene-labeled actin monomers was initiated with 3 nM gelsolin-actin dimers in the presence of 0–580 nM Arp2/3 complex as indicated. (Inset) Time courses of pointed-end elongation monitored by pyrene fluorescence. (B) Inhibition of depolymerization of gelsolin-capped actin filaments by Arp2/3 complex. Pyrene-labeled (10%) actin filaments (20 μM), assembled from 0.03 μM gelsolin-actin nuclei, were diluted to 0.2 μM (<pointed end critical concentration) in Arp2/3 concentrations from 0 to 340 nM. (Inset) Pyrene fluorescence traces in the absence (leftmost trace) and presence of increasing concentrations of Arp2/3 complex (progressively slower traces). (C) Effect of Arp2/3 complex on the critical concentration for actin polymerization. A range of concentrations of pyrene-labeled actin (7%) were assembled to steady-state spontaneously (○, •) or from 3 nM gelsolin-actin seeds (▵, ▴) in the absence (open symbols) or presence (closed symbols) of 0.3 μM Arp2/3 complex. The extent of polymerization was measured by pyrene fluorescence after 14 h. We measured critical concentration from the intersections of the filamentous actin fluorescence with actin monomer fluorescence (⧫). Conditions: temperature: 24°C; buffer: 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, and 10 mM imidazole (pH 7.0).

Figure 2

Arp2/3 complex nucleates actin filament polymerization. (A) Spontaneous polymerization of 5.3 μM pyrene-labeled (7%) Acanthamoeba actin is accelerated by Arp2/3 complex. Conditions: same as Fig. 1. The concentrations of complex in the samples are, from right to left, 0 μM, 0.037 μM, 0.15 μM, 0.58 μM, and 2.3 μM. Solid lines are kinetic simulations based on binding of Arp2/3 complex to actin dimers and the rate constants in Table 2. (Inset) Early time points of polymerization. Conditions: same as Fig. 1. (B) Models for actin filament nucleation by Arp2/3 complex: Arp2/3 complex interacts sequentially with two actin monomers (m), with an actin dimer (d), or with a trimer (t) to form a nucleus for filament elongation.

Figure 3

Electron micrographs of quick-frozen, deep-etched, and rotary-shadowed samples of Arp2/3 complex (A) and complex mixed with gelsolin-capped actin filaments (B-D). In the presence of Arp2/3, complex actin filaments form branching arbors with numerous end-to-side connections between filaments (B). The branch points appear to be rigid attachments with a fixed 70° angle between actin filaments (C) and frequently contain a globular mass at the point of attachment (C, left arrow in B). (D) Filaments partially decorated with Arp2/3 complex. (E) Globular masses associated with filament pointed ends in the presence of Arp2/3 complex. Conditions: buffer same as Fig. 1.

Figure 4

Dendritic nucleation model for actin polymerization, capping, and network formation at the leading edge of a motile cell. Inactive or sequestered Arp2/3 complex (a) is recruited to the leading edge of the cell by an unknown mechanism (b). The complex nucleates new actin filaments either while free (c) or while bound to the side of existing filaments (d). The complex anchors the branch at a fixed angle of 70° relative to the barbed ends of the two filaments. Rapid growth of new filaments in the barbed direction, fed by actin subunits from the actin-profilin pool, expands the actin filament network. Subsequent disassembly of the network deep in the cytoplasm regenerates subunits for later growth of the cortical network.