© 2000 Macmillan Magazines Ltd articles NATURE CELL BIOLOGY | VOL 2 | JULY 2000 | www.nature.com/ncb 385 The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins Dominique Pantaloni*, Rajaa Boujemaa*, Dominique Didry*, Pierre Gounon† and Marie-France Carlier*‡ *Laboratoire d’Enzymologie et Biochimie Structurale, CNRS, 91198 Gif-sur-Yvette, France †Microscopie Electronique, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris, France ‡e-mail: carlier@lebs.cnrs-gif.fr The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility. ctin polymerization in response to signalling drives the extension of lamellipodia, the formation of microspikes and the propulsion of Listeria and Shigella. Reconstitution of actin-based motility from a minimum set of pure proteins 1 (reviewed in ref. 2) has provided insights into the mechanism by which actin polymerization generates movement. Filaments are generated at the leading edges or the surfaces of Listeria and Shigella 3,4 by the Arp2/3 complex. They are then locally activated, by the protein ActA at the surface of Listeria 5 , and by proteins of the Wiskott–Aldrich syndrome protein (WASP) family, which connect signalling to actin assembly, in eukaryotic cells 6–10 and at the surface of Shigella 11 . Sustained movement results from rapid, ATP-driven treadmilling of actin filaments 12 , mediated by the combined activi- ties of actin depolymerizing factor and capping proteins 13 . Force production is restricted to specific sites as a result of the ‘decommis- sioning’ action of capping proteins, which prevent growth of fila- ments that are disconnected from the growth site. In lamellipodia, actin filaments are organized in a Y-shaped, branched array, and Arp2/3 is localized at the branches 14 . Branched filaments can also be observed, by electron microscopy, in solutions of actin polymerized in the presence of the Arp2/3 complex 15 . In Shigella-infected cells, N-WASP localizes exclusively to the bacterial surface, whereas Arp2/3 is present throughout the actin tail 11 . In vitro studies of the effect of Arp2/3 on actin have shown that the Arp2/3 complex by itself is a poor nucleator, but, when acti- vated by the isolated carboxy-terminal WA domain of WASP/Scar proteins, it greatly stimulates actin polymerization. Activation of Arp2/3 implies binding of WA to both Arp2/3 and G-actin 6 . The WA–actin complex associates productively with barbed filament ends 11 . The finding that preformed actin filaments further A Figure 1 WA-activated Arp2/3 stimulates actin polymerization in an autocatalytic manner. a, MgATP–G-actin (2.5 μM, 10%, pyrenyl-labelled) was polymerized in the presence of 0.6 μM WA and the Arp2/3 complex at the following concentrations (right to left): 0, 1.76, 3.52, 7.2, 14.3, 28.5, 56, 71 nM. Noisy lines are pyrene-fluorescence measurements converted to μM F-actin. Dotted lines are calculated time courses according to the model presented in the text. Inset, plot of the recorded time courses in the presence of Arp2/3 against time. The mid- polymerization time was taken as the time origin for each curve. Heavy continuous and dashed curves correspond to the nucleation–growth process observed in the absence of Arp2/3, plotted on the same time scale and a 20-fold-compressed time scale, respectively. b, Kinetic analysis of the polymerization curve for 7.1 nM Arp2/ 3 in a, showing time dependences of the mass of F-actin (diamonds), its time derivative (dotted line), concentration of filament ends ([F], continuous line) and average filament length (inset). Note that, between t = 300 s and t = 850 s, the average length varies from 5–8.5μm, whereas, in the same time interval, the number of filament ends increases tenfold. Time (s) 0 0.5 1 1.5 2 2.5 0 500 1,000 1,500 2,000 Polymerized-actin concentration (μM) Polymerized-actin concentration (μM) 0 1 2 0 0.2 0.4 0.6 0 300 600 900 1,200 1,500 [F] (nM) Time (s) Time (s) 5 10 300 600 b a –2 –1 0 1 –200 0 200 Time (s) ln{(Φ – Φ o )/(Φ max – Φ)} <l > (μm)