Motile cells extend a leading edge by assembling a branched network of actin filaments that produces physical force as the polymers grow beneath the plasma membrane. A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion. Signaling pathways converging on WASp/Scar proteins regulate the activity of Arp2/3 complex, which mediates the initiation of new filaments as branches on preexisting filaments. After a brief spurt of growth, capping protein terminates the elongation of the filaments. After filaments have aged by hydrolysis of their bound ATP and dissociation of the gamma phosphate, ADF/cofilin proteins promote debranching and depolymerization. Profilin catalyzes the exchange of ADP for ATP, refilling the pool of ATP-actin monomers bound to profilin, ready for elongation.

Summary The protein actin forms filaments that provide cells with mechanical support and driving forces for movement. Actin contributes to biological processes such as sensing environmental forces, internalizing membrane vesicles, moving over surfaces and dividing the cell in two. These cellular activities are complex; they depend on interactions of actin monomers and filaments with numerous other proteins. Here, we present a summary of the key questions in the field and suggest how those questions might be answered. Understanding actin-based biological phenomena will depend on identifying the participating molecules and defining their molecular mechanisms. Comparisons of quantitative measurements of reactions in live cells with computer simulations of mathematical models will also help generate meaningful insights.

We review how motile cells regulate actin filament assembly at their leading edge. Activation of cell surface receptors generates signals (including activated Rho family GTPases) that converge on integrating proteins of the WASp family (WASp, N-WASP, and Scar/WAVE). WASP family proteins stimulate Arp2/3 complex to nucleate actin filaments, which grow at a fixed 70 degrees angle from the side of pre-existing actin filaments. These filaments push the membrane forward as they grow at their barbed ends. Arp2/3 complex is incorporated into the network, and new filaments are capped rapidly, so that activated Arp2/3 complex must be supplied continuously to keep the network growing. Hydrolysis of ATP bound to polymerized actin followed by phosphate dissociation marks older filaments for depolymerization by ADF/cofilins. Profilin catalyzes exchange of ADP for ATP, recycling actin back to a pool of unpolymerized monomers bound to profilin and thymosin-beta 4 that is poised for rapid elongation of new barbed ends.

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.

and Developmental Biology time, provided a coherent, semi-quantitative model for Yale University the molecular mechanism of protrusion of lamellae and New Haven, Connecticut 06520 how cells might respond to external signals. 2 Department of Cellular and Molecular Biology Actin filaments are, by mass, the dominant structural Northwestern University component of the lamellipodium and, indeed, actin is School of Medicine the most abundant protein in many eukaryotic cells. Chicago, Illinois 60611The filaments are double helical polymers of globular subunits all arranged head-to-tail to give the filament a molecular polarity (Figure 2). Based on the arrowhead pattern created by decoration with myosin, one end is Motile cells extend a leading edge by assembling a called the barbed end and the other the pointed end. branched network of actin filaments that producesThis polarity is key to the mechanism of actin assembly physical force as the polymers grow beneath the in cells. The barbed end is favored for growth and actin plasma membrane. A core set of proteins including filaments in cells are strongly oriented with respect to actin, Arp2/3 complex, profilin, capping protein, and the cell surface, barbed ends outward (Small et al., ADF/cofilin can reconstitute the process in vitro, and 1978). Accordingly, when permeabilized cells are promathematical models of the constituent reactions previded with fluorescent actin subunits, they add to dict the rate of motion. Signaling pathways converging barbed ends at the leading edge of the lamellum (Syon WASp/Scar proteins regulate the activity of Arp2/3 mons and Mitchison, 1991; Chan et al., 2000). complex, which mediates the initiation of new fila-Marking experiments, by photoactivating caged-fluoments as branches on preexisting filaments. After a rescent actin (Theriot and Mitchison, 1991) showed that brief spurt of growth, capping protein terminates the in fast moving cells, like fish epidermal keratocytes, actin elongation of the filaments. After filaments have aged filaments remain stationary while the cell advances, thus by hydrolysis of their bound ATP and dissociation of demonstrating that protrusion of the leading edge octhe ␥ phosphate, ADF/cofilin proteins promote debcurs concomitantly with polymerization of actin. Alternaranching and depolymerization. Profilin catalyzes the tively, if a cell is stationary, like disc-shaped sea urchin exchange of ADP for ATP, refilling the pool of ATPcoelomocytes, actin filaments assemble at the margin actin monomers bound to profilin, ready for elonof the cell and move away from the edge (Wang, 1985; gation. Edds, 1993; Henson et al., 1999), reflecting the same relationship to the cell surface as in locomotion. More commonly, as in fibroblasts, actin polymerization is Directional motility is a fundamental cellular process transformed partially into protrusion and partially into essential for embryonic development, wound healing, retrograde flow (Theriot and Mitchison, 1992; Lin and immune responses, and development of tissues. ForFor...

This review summarizes what is known about the biochemical and biophysical mechanisms that initiate the assembly of actin filaments in cells. Assembly and disassembly of these filaments contribute to many types of cellular movements. Numerous proteins regulate actin assembly, but Arp2/3 complex and formins are the focus of this review because more is known about them than other proteins that stimulate the formation of new filaments. Arp2/3 complex is active at the leading edge of motile cells, where it produces branches on the sides of existing filaments. Growth of these filaments produces force to protrude the membrane. Crystal structures, reconstructions from electron micrographs, and biophysical experiments have started to map out the steps through which proteins called nucleation-promoting factors stimulate the formation of branches. Formins nucleate and support the elongation of unbranched actin filaments for cytokinesis and various types of actin filament bundles. Formins associate processively with the fast-growing ends of filaments and protect them from capping.

We used fluorescence microscopy to measure global and local concentrations of 28 cytoskeletal and signaling proteins fused to yellow fluorescent protein (YFP) in the fission yeast Schizosaccharomyces pombe. Native promoters controlled the expression of these functional YFP fusion proteins. Fluorescence measured by microscopy or flow cytometry was directly proportional to protein concentration measured by quantitative immunoblotting. Global cytoplasmic concentrations ranged from 0.04 (formin Cdc12p) to 63 micromolar (actin). Proteins concentrated up to 100 times in contractile rings and 7500 times in spindle pole bodies at certain times in the cell cycle. This approach can be used to measure the global and local concentrations of any fusion protein.

Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.