3 Complex

Cell, Vol. 107, 703–705, December 14, 2001, Copyright 2001 by Cell Press

Arp2/3 Complex: Advances on the Inner Workings of a Molecular Machine John A. Cooper,1,2 Martin A. Wear, and Alissa M. Weaver Department of Cell Biology Washington University School of Medicine Saint Louis, Missouri 63130

Several new papers report progress on the structure and function of Arp2/3 complex. A crystal structure, a cryo-EM structure, and a reconstitution of the complex from subunits have been reported. New results also address the nucleation mechanism and the role of bound nucleotide. Our understanding of how actin polymerization contributes to cell motility has taken giant strides in recent years based on the discovery of the Arp2/3 complex. The extension of a cell process is caused by polymerization of actin filaments that push the plasma membrane forward. Many cell processes, such as lamella and lamellipodia, are composed of highly branched networks of actin filaments, with their barbed ends (the ends favored for polymerization) oriented toward the plasma membrane at the cell’s periphery. Many network branch points, ones between the side of one actin filament and the pointed end of another filament, contain an Arp2/3 complex molecule (Borisy and Svitkina, 2000). Arp2/3 binds to the side of an existing filament (the “mother” filament) and nucleates the formation of a new filament (the “daughter” filament) with a free barbed end. Actin subunits then add to the new free barbed end, pushing the membrane outward. Before the discovery of Arp2/3 complex, we knew that the cortex of cells had branching networks of actin filaments, but we had few appealing biochemical hypotheses for what mediated end-to-side branches or what created free barbed ends. Arp2/3 now appears to be the explanation for both, at least in many cell types and situations (Borisy and Svitkina, 2000). The actin assembly field has now become heavily focused on Arp2/3’s structure, function, and regulation. Meetings last summer, including the motility Gordon Conference, featured a large number of exciting discoveries, which are now being published. This minireview will highlight a few of these exciting new advances. Structure of Arp2/3 Complex A high resolution (2.0 A˚) crystal structure for Arp2/3 complex has been reported by Pollard and colleagues (Robinson et al., 2001) (Figure 1A). Arp2/3 complex consists of seven subunits—two actin-related proteins, Arp2 and Arp3, along with five unique polypeptides, called ARPC1-5 (Table 1). The Arp2 and Arp3 polypeptides are folded like actin, as expected from previous modeling studies based on their sequences (Kelleher et al., 1995). ARPC1/p40 is a ␤-propeller protein with seven blades, also predicted from its sequence. ARPC2/p34 1 2

Correspondence: [email protected] The writing of this article was funded by NIH grant GM 38542.

Minireview

and ARPC4/p20 have large amounts of buried surface area and are predicted to form a heterodimer at the core of the complex (Figure 1A). This prediction is borne out by reconstitution studies from Welch and colleagues (Gournier et al., 2001), discussed below. ARPC1/p40 and ARPC3/p21 have large basic patches on their exposed surfaces, which may be important for binding the acidic domains of activators (Figure 1B). The subunit interactions are largely consistent with previous data from chemical crosslinking and two-hybrid studies (Mullins and Pollard, 1999). The structure corresponds to the inactive form of Arp2/3 complex. Alone, Arp2/3 has little or no activity. It can be activated by WASp/Scar/WAVE family members, Listeria ActA, cortactin, budding yeast Abp1 and Pan1, fission yeast myosin-I’s, and Dictyostelium CARMIL proteins (Duncan et al., 2001; Goode et al., 2001; Higgs and Pollard, 2001; Jung et al., 2001). Most of these proteins bind Arp2/3 via a conserved acidic domain. The binding of Arp2/3 to the side of a mother actin filament also activates Arp2/3 complex, in a manner cooperative with the activation by WASps (Higgs and Pollard, 2001). In the structure of the inactive complex, the arrangement of Arp2 and Arp3 is not consistent with their binding conventional actin subunits to nucleate a new filament with a free barbed end (Robinson et al., 2001). One must presume that a substantial conformational change occurs upon activation of the complex, so that actin subunits add to Arp2 and Arp3 to form the daughter filament. Pollard and colleagues propose such a possible conformational change (Robinson et al., 2001). An important next step will be to determine a crystal structure for active Arp2/3. The crystal structure can now be compared with a low resolution (32 A˚) cryo-EM structural reconstruction of WASp-WA-activated Arp2/3 complex in association with actin filaments, recently published by Hanein and colleagues (Volkmann et al., 2001). At present, the gross topological outline of the whole Arp2/3 complex can be fit into a density profile observed on the EM images (Figure 2), but the resolution of the reconstruction does not allow one to definitively identify the individual polypeptides, with the possible exception of Arp2 and Arp3. In the proposed model, Arp2/3 complex sits along the side of the mother actin filament, not intercalating in any gross way into its helical structure. Arp2/3 complex appears to contact the mother actin filament at three positions. Based on previous chemical crosslinking, two-hybrid, and genetic experiments, Hanein and colleagues propose that the ARPC1/p40, ARPC2/p35, and ARPC5/p14 subunits of Arp2/3 contact the side of the mother filament, and that the Arp2 and Arp3 subunits contact the pointed end of the daughter filament (Volkmann et al., 2001). Hanein and colleagues were able to determine a cryo-EM structure from WASP-WA-complexed Arp2/3 but not inactive Arp2/3, supporting the idea that activators induce a substantial conformational change in Arp2/3 complex.

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Figure 1. Crystal Structure of Arp2/3 Complex Modified, with permission, from Robinson et al. (2001); figure kindly provided by Bob Robinson, Kursi Turbedsky, and Tom Pollard. (A) Ribbon diagram with the individual subunits colored as indicated. The lightly colored portion of Arp2 in (A), which is outlined in (B), was not resolved in the crystal structure and was modeled from the crystal structure of actin. (B) Space filling model with the electrostatic potential indicated with blue as positive and red as negative.

Reconstitution of Arp2/3 Complex The entire Arp2/3 complex has been reconstituted by Welch and colleagues, who simultaneously expressed all seven subunits in a baculovirus/insect cell expression system (Gournier et al., 2001). The reconstituted complete complex has the same biochemical activity seen with native Arp2/3 complex, purified from tissue, showing that substoichiometric or trace amounts of other components are probably not important. The reconstitution system allows one to address a number of specific issues about how the complex is assembled and functions. In their initial work, Welch and colleagues expressed different subunits in combination, omitting one or more (Gournier et al., 2001). One interesting result was that the ARPC2/p34 and ARPC4/p20 subunits bind tightly to each other in the absence of other subunits, and they are each necessary for the assembly of the complex, indicating that this heterodimer functions as a structural core for the complex. The arrangement of the subunits in the crystal structure supports this conclusion (Robinson et al., 2001). In terms of functional activity, the presence of Arp3 is necessary for the complex to nucleate, which supports the notion that Arp2 and Arp3 create a surface resembling the barbed end of an actin filament. Further-

more, the heterodimer of ARPC2/p34 and ARPC4/p20 alone binds to the sides of filaments with an affinity similar to that of the whole Arp2/3 complex. In previous work, antibodies to ARPC2/p34 prevented Arp2/3 complex from binding to the side of an actin filament. These antibodies inhibited nucleation and branching, confirming the coupling of the side binding and nucleation activities (Bailly et al., 2001). ARPC2/p34 is predicted to contact the side of the mother actin filament in the cryo-EM model (Volkmann et al., 2001). On the other hand, ARPC1/p40

Table 1. Nomenclature for the Arp2/3 Complex Subunits Standarized

Mammal

Acanthamoeba

Saccharomyces

ARPC1 ARPC2 ARPC3 ARPC4 ARPC5

p41/p40 p34 p21 p20 p16

p40 p35 p18 p19 p14

Arc40p Arc35p Arc18p Arc19p Arc15p

Arp2 and Arp3 have the same name in all systems. The subunit names for the Dictyostelium polypeptides are the same as those for mammals.

Figure 2. Model for the Structure of Arp2/3 Complex at an End-ToSide Actin Filament Branch Point This model was proposed by Hanein and colleagues, based on their cryo-EM results. Modified, with permission, from Volkmann et al. (2001); figure kindly provided by Niels Volkmann and Dorit Hanein.

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and ARPC5/p14, which are predicted to contact the mother actin filament in the cryo-EM model (Volkmann et al., 2001), were not important for branch formation in the reconstitution experiments (Gournier et al., 2001). Conversely, the ARPC3/p21 subunit did contribute to the efficiency of branching. Thus, at present, one can conclude that ARPC2/p34 is necessary for side binding and branch formation, while the role of the other subunits remains to be determined. In the future, the reconstitution system should allow one to test the effect of more subtle mutations and thereby determine with greater certainty how Arp2/3 complex is assembled, functions, and is activated. Function—End Versus Side Binding The field has been in the throes of a controversy about whether Arp2/3 prefers to bind to the sides or the barbed ends of the mother filament. There now seems to be some level of agreement that Arp2/3 binds to the sides of actin filaments but prefers to bind near growing barbed ends, which contain ATP. Pollard’s group found that Arp2/3 could bind to the sides of actin filaments in their initial characterization of the complex (Mullins et al., 1997). They then used light microscopy of fluorescent phalloidin-stained filaments, which can distinguish old from new filaments, to show that new filaments (nucleated by Arp2/3) could grow from the sides of old filaments (Amann and Pollard, 2001a). On the other hand, the Carlier lab had observations based on the length and history of branching of the networks, combined with modeling of the time course of polymerization under different conditions, which argued that Arp2/3 complex nucleated only from the barbed end of a mother filament (Pantaloni et al., 2000). Carlier and colleagues even considered a model in which Arp2/3 added to the barbed end, with one Arp intercalating itself into the structure of the mother filament in place of one conventional actin subunit. While the cryo-EM structural results appear to rule out Arp2/3 complex adding directly to the barbed end, new observations from the Condeelis lab and the Pollard lab appear to resolve the issue and show that both models are correct in different ways (Amann and Pollard, 2001b; Ichetovkin et al., 2001). Condeelis and colleagues visualized the assembly of actin filament networks, using red and green fluorescent phalloidin to distinguish new from old actin filaments (Ichetovkin et al., 2001). These data show that daughter filaments, nucleated by Arp2/3, can form from the sides of mother filaments; however, the preferred site of nucleation is close to the barbed end of a growing mother filament. These results were also supported by movies of growing actin filaments. An attractive interpretation of these results is that Arp2/3 prefers to bind to the sides of actin filaments that contain subunits with ATP. Free actin subunits bind ATP, which hydrolyzes to ADP over time after the subunits add to the barbed end. Thus, a preference for branching from ATP-F-actin would be revealed as a bias for branching near the barbed end of a growing filament. Additional experiments with other adenosine nucleotides and a nonhydrolysable ATP analog support this hypothesis (Ichetovkin et al., 2001). Amann and Pollard also collected movies of growing actin filaments and found a preference for branching near barbed ends, under conditions where the barbed ends should have a cap of ATP-containing subunits

(Amann and Pollard, 2001b). In the absence of ATP caps, the preference was lost. Function—Nucleotide Content and Binding Arp2 and Arp3 bind an adenosine nucleotide, preferably ATP, as does actin. The potential role of this nucleotide in regulating the activity and conformation of Arp2/3 complex is an important question. The Arp2/3 crystal structure does not include any bound nucleotide. Recent papers from the labs of Carlier and Mullins address the nature and significance of the nucleotide (Dayel et al., 2001; Le Clainche et al., 2001). Both groups find that bound ATP is important for the nucleation activity of Arp2/3 complex, and that ATP binding affinity is increased upon activation. Neither group finds evidence that bound ATP hydrolyzes. There was also some disagreement between the two groups as to the affinities of nucleotide binding and the differential selectivity of Arp2 and Arp3 for ATP binding. However, the binding of adenosine nucleotide—preferentially hydrolysable ATP—appears be important for the complex to nucleate efficiently. In summary, substantial progress has been made in determining the structure of Arp2/3 complex and how interactions with actin filaments, ATP, and WASp family activators may induce activation of the complex. Selected Reading Amann, K.J., and Pollard, T.D. (2001a). Nat. Cell Biol. 3, 306–310. Amann, K.J., and Pollard, T.D. (2001b). Proc. Natl. Acad. Sci. USA 98, 15009–15013. Bailly, M., Ichetovkin, I., Grant, W., Zebda, N., Machesky, L.M., Segall, J.E., and Condeelis, J. (2001). Curr. Biol. 11, 620–625. Borisy, G.G., and Svitkina, T.M. (2000). Curr. Opin. Cell Biol. 12, 104–112. Dayel, M.J., Holleran, E.A., and Mullins, R.D. (2001). Proc. Natl. Acad. Sci. USA 98, 14871–14876. Duncan, M.C., Cope, M.J., Goode, B.L., Wendland, B., and Drubin, D.G. (2001). Nat. Cell Biol. 3, 687–690. Goode, B.L., Rodal, A.A., Barnes, G., and Drubin, D.G. (2001). J. Cell Biol. 153, 627–634. Gournier, H., Goley, E.D., Niederstrasser, H., Trinh, T., and Welch, M.D. (2001). Mol. Cell 8, 1041–1052. Higgs, H.N., and Pollard, T.D. (2001). Annu. Rev. Biochem. 70, 649–676. Ichetovkin, I., Grant, W., and Condeelis, J. (2001). Curr. Biol., in press. Jung, G., Remmert, K., Wu, X., Volosky, J.M., and Hammer, J.A., 3rd. (2001). J. Cell Biol. 153, 1479–1497. Kelleher, J.F., Atkinson, S.J., and Pollard, T.D. (1995). J. Cell Biol. 131, 385–397. Le Clainche, C., Didry, D., Carlier, M.F., and Pantaloni, D. (2001). J. Biol. Chem. Published online October 11, 2001. 10.1074/jbc. C100476200. Mullins, R.D., and Pollard, T.D. (1999). Curr. Opin. Struct. Biol. 9, 244–249. Mullins, R.D., Stafford, W.F., and Pollard, T.D. (1997). J. Cell Biol. 136, 331–343. Pantaloni, D., Boujemaa, R., Didry, D., Gounon, P., and Carlier, M.F. (2000). Nat. Cell Biol. 2, 385–391. Robinson, R.C., Turbedsky, K., Kaiser, D.A., Marchand, J.-B., Higgs, H.N., Choe, S., and Pollard, T.D. (2001). Science 294, 1679–1684. Volkmann, N., Amann, K.J., Stoilova-McPhie, S., Egile, C., Winter, D.C., Hazelwood, L., Heuser, J.E., Li, R., Pollard, T.D., and Hanein, D. (2001). Science 293, 2456–2459.