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Follow the Monomer

2008; Cell Press; Volume: 133; Issue: 5 Linguagem: Inglês

10.1016/j.cell.2008.05.012

1097-4172

James E. Bear,

Skin and Cellular Biology Research

Capping proteins limit actin filament growth, but paradoxically increase actin-based cell motility. This has been attributed to funneling of actin monomers to the filament ends that remain uncapped. Using a reconstituted motility system, Akin and Mullins, 2008Akin O. Mullins R.D. Cell. 2008; (this issue)PubMed Google Scholar now demonstrate that filament capping increases Arp2/3-based nucleation and branching, rather than elevating the rate of filament elongation. Capping proteins limit actin filament growth, but paradoxically increase actin-based cell motility. This has been attributed to funneling of actin monomers to the filament ends that remain uncapped. Using a reconstituted motility system, Akin and Mullins, 2008Akin O. Mullins R.D. Cell. 2008; (this issue)PubMed Google Scholar now demonstrate that filament capping increases Arp2/3-based nucleation and branching, rather than elevating the rate of filament elongation. Although actin can polymerize into filaments on its own, cells use an army of proteins to control the starting and stopping of this reaction, as well as to organize the filaments into useful structures. Studying the function and regulation of these actin-modulating proteins has been the work of many labs for several decades. The control of actin dynamics is not a mere academic curiosity, but plays a key role in physiological processes such as morphogenesis and immune system function as well as in diseases such as metastatic cancer. Two important factors for controlling actin dynamics are the Arp2/3 complex, which nucleates new filaments and concomitantly anchors them to the sides of existing filaments, and the capping protein, which binds to the rapidly growing barbed ends of filaments and terminates their growth (reviewed in Pollard and Borisy, 2003Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3267) Google Scholar). Although these factors have been extensively studied in vitro, the relationship between their biochemical activities and their effects on motility is complex. This is particularly true of capping proteins that block filament growth yet enhance cell motility in vitro and in vivo (van der Gucht et al., 2005van der Gucht J. Paluch E. Plastino J. Sykes C. Proc. Natl. Acad. Sci. USA. 2005; 102: 7847-7852Crossref PubMed Scopus (112) Google Scholar, Hug et al., 1995Hug C. Jay P.Y. Reddy I. McNally J.G. Bridgman P.C. Elson E.L. Cooper J.A. Cell. 1995; 81: 591-600Abstract Full Text PDF PubMed Scopus (146) Google Scholar). One explanation for this paradoxical set of observations is the “actin funneling hypothesis,” which posits that capping proteins enhance motility by capping most actin filaments in the reaction and funneling the increased number of free actin monomers onto a small subset of filaments that grow with higher rates of elongation (Carlier and Pantaloni, 1997Carlier M.F. Pantaloni D. J. Mol. Biol. 1997; 269: 459-467Crossref PubMed Scopus (254) Google Scholar) (Figure 1A). In their new study, Akin and Mullins, 2008Akin O. Mullins R.D. Cell. 2008; (this issue)PubMed Google Scholar re-examine this question and come to a strikingly different conclusion about the role of capping protein in enhancing motility. They used an established in vitro motility system (Loisel et al., 1999Loisel T.P. Boujemaa R. Pantaloni D. Carlier M.F. Nature. 1999; 401: 613-616Crossref PubMed Scopus (801) Google Scholar) comprising polystyrene beads coated with the Arp2/3-activator ActA. These beads were incubated in a precise mixture of purified protein components including nonmuscle actin, the Arp2/3 complex, capping protein, cofilin, and profilin. This system allowed them to visualize the initiation of actin assembly that occurs in a shell around the beads and the subsequent symmetry breaking event that leads to sustained movement of the beads on rocket tails of branched, polymerizing actin filaments. Using this approach, they systematically varied the concentration of the Arp2/3 complex and capping protein to test several tenets of the actin funneling hypothesis. Consistent with previous work, they found that an increase in capping protein increased the rate of bead motility. One clear prediction of the actin funneling hypothesis is that increasing capping protein should increase the concentration of actin monomers; this in turn should lead to faster rates of filament elongation on the few uncapped filaments in the reaction. On the contrary, increasing capping protein had no effect on actin monomer concentration in these reactions despite the clear increase in bead movement. Two other predictions of the actin funneling hypothesis are that the rate of filament growth on the bead surface should increase and that the number of filaments contributing to motility will decrease with increased concentration of capping protein. Again, the rate of filament elongation and the number of filaments were unaffected by increased capping protein. So why does increasing the capping protein concentration lead to increased motility? The authors postulate an intriguing alternate hypothesis: Increased capping protein leads to enhanced nucleation mediated by Arp2/3. In their experiments, increasing the capping protein concentration led to an increased number of capped filaments, as expected. However, the total number of filaments remained constant, suggesting that increased nucleation must be occurring. Indeed, the ratio of the Arp2/3 complex to actin increases with higher levels of capping protein, indicating that more nucleation is happening. This effect arises due to higher local availability of actin monomers at the bead surface when more filaments are capped. Given that actin monomers are essential for Arp2/3-based nucleation, more monomers means more nucleation rather than increased barbed-end elongation. The authors term this the “monomer gating model” (Figure 1C). This notion of momomer gating has important implications for interpreting some previous studies and will influence future work in this area. Factors such as the Ena/VASP proteins that inhibit capping of actin filaments though an anticapping effect would be predicted to indirectly decrease Arp2/3 branching. Indeed, this is entirely consistent with studies where Ena/VASP proteins are targeted to the plasma membrane or added to in vitro reconstitution systems and branch frequency is reduced (Figure 1B) (Bear et al., 2002Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, Samarin et al., 2003Samarin S. Romero S. Kocks C. Didry D. Pantaloni D. Carlier M.F. J. Cell Biol. 2003; 163: 131-142Crossref PubMed Scopus (122) Google Scholar). It will be interesting to see if other anticapping proteins such as formins also decrease branching in reconstitution systems that include Arp2/3 and capping protein. Although this study breaks important new ground, it will be useful to further verify this effect in the more complex environment found inside cells. A key experiment will be to analyze the effect of inositol phospholipids, such as PIP2, on this process given that they regulate both Arp2/3-activating proteins (such as N-WASP) and capping protein. It will also be exciting to see these ideas incorporated into future biophysical models of actin polymerization-induced force generation. We thank L. Cai for help with the figure. Capping Protein Increases the Rate of Actin-Based Motility by Promoting Filament Nucleation by the Arp2/3 ComplexAkin et al.CellMay 30, 2008In BriefCapping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. Full-Text PDF Open Archive