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• W2023808883 abstract "Cell locomotion and endocytosis are powered by the rapid polymerization and turnover of branched actin filament networks nucleated by Arp2/3 complex [1Goley E.D. Welch M.D. The ARP2/3 complex: An actin nucleator comes of age.Nat. Rev. Mol. Cell Biol. 2006; 7: 713-726Crossref PubMed Scopus (623) Google Scholar]. Although a large number of cellular factors have been identified that stimulate Arp2/3 complex-mediated actin nucleation, only a small number of studies so far have addressed which factors promote actin network debranching [2Blanchoin L. Pollard T.D. Mullins R.D. Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks.Curr. Biol. 2000; 10: 1273-1282Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Cai L. Makhov A.M. Schafer D.A. Bear J.E. Coronin 1B antagonizes cortactin and remodels Arp2/3-containing actin branches in lamellipodia.Cell. 2008; 134: 828-842Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 4Chan C. Beltzner C.C. Pollard T.D. Cofilin dissociates Arp2/3 complex and branches from actin filaments.Curr. Biol. 2009; 19: 537-545Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. Here, we investigated the function of a conserved homolog of ADF/cofilin, glia maturation factor (GMF) [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar, 6Goroncy A.K. Koshiba S. Tochio N. Tomizawa T. Sato M. Inoue M. Watanabe S. Hayashizaki Y. Tanaka A. Kigawa T. Yokoyama S. NMR solution structures of actin depolymerizing factor homology domains.Protein Sci. 2009; 18: 2384-2392Crossref PubMed Scopus (34) Google Scholar]. We found that S. cerevisiae GMF (also called Aim7) localizes in vivo to cortical actin patches and displays synthetic genetic interactions with ADF/cofilin. However, GMF lacks detectable actin binding or severing activity and instead binds tightly to Arp2/3 complex. Using in vitro evanescent wave microscopy, we demonstrated that GMF potently stimulates debranching of actin filaments produced by Arp2/3 complex. Further, GMF inhibits nucleation of new daughter filaments. Together, these data suggest that GMF binds Arp2/3 complex to both “prune” daughter filaments at the branch points and inhibit new actin assembly. These activities and its genetic interaction with ADF/cofilin support a role for GMF in promoting the remodeling and/or disassembly of branched networks. Therefore, ADF/cofilin and GMF, members of the same superfamily, appear to have evolved to interact with actin and actin-related proteins, respectively, and to make mechanistically distinct contributions to the remodeling of cortical actin structures. Cell locomotion and endocytosis are powered by the rapid polymerization and turnover of branched actin filament networks nucleated by Arp2/3 complex [1Goley E.D. Welch M.D. The ARP2/3 complex: An actin nucleator comes of age.Nat. Rev. Mol. Cell Biol. 2006; 7: 713-726Crossref PubMed Scopus (623) Google Scholar]. Although a large number of cellular factors have been identified that stimulate Arp2/3 complex-mediated actin nucleation, only a small number of studies so far have addressed which factors promote actin network debranching [2Blanchoin L. Pollard T.D. Mullins R.D. Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks.Curr. Biol. 2000; 10: 1273-1282Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 3Cai L. Makhov A.M. Schafer D.A. Bear J.E. Coronin 1B antagonizes cortactin and remodels Arp2/3-containing actin branches in lamellipodia.Cell. 2008; 134: 828-842Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 4Chan C. Beltzner C.C. Pollard T.D. Cofilin dissociates Arp2/3 complex and branches from actin filaments.Curr. Biol. 2009; 19: 537-545Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. Here, we investigated the function of a conserved homolog of ADF/cofilin, glia maturation factor (GMF) [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar, 6Goroncy A.K. Koshiba S. Tochio N. Tomizawa T. Sato M. Inoue M. Watanabe S. Hayashizaki Y. Tanaka A. Kigawa T. Yokoyama S. NMR solution structures of actin depolymerizing factor homology domains.Protein Sci. 2009; 18: 2384-2392Crossref PubMed Scopus (34) Google Scholar]. We found that S. cerevisiae GMF (also called Aim7) localizes in vivo to cortical actin patches and displays synthetic genetic interactions with ADF/cofilin. However, GMF lacks detectable actin binding or severing activity and instead binds tightly to Arp2/3 complex. Using in vitro evanescent wave microscopy, we demonstrated that GMF potently stimulates debranching of actin filaments produced by Arp2/3 complex. Further, GMF inhibits nucleation of new daughter filaments. Together, these data suggest that GMF binds Arp2/3 complex to both “prune” daughter filaments at the branch points and inhibit new actin assembly. These activities and its genetic interaction with ADF/cofilin support a role for GMF in promoting the remodeling and/or disassembly of branched networks. Therefore, ADF/cofilin and GMF, members of the same superfamily, appear to have evolved to interact with actin and actin-related proteins, respectively, and to make mechanistically distinct contributions to the remodeling of cortical actin structures. Yeast GMF localizes to actin patches and genetically interacts with ADF/cofilin GMF binds to Arp2/3 complex but not actin GMF inhibits actin nucleation and stimulates filament debranching GMF and cofilin use distinct mechanisms to remodel dendritic actin networks Glia maturation factor (GMF) is an evolutionarily conserved member of the ADF/cofilin superfamily based on sequence comparisons and structural homology [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar, 6Goroncy A.K. Koshiba S. Tochio N. Tomizawa T. Sato M. Inoue M. Watanabe S. Hayashizaki Y. Tanaka A. Kigawa T. Yokoyama S. NMR solution structures of actin depolymerizing factor homology domains.Protein Sci. 2009; 18: 2384-2392Crossref PubMed Scopus (34) Google Scholar]. Sequence alignments and three-dimensional structure comparisons suggest that although many general features of the ADF/cofilin fold are retained in GMF-γ, the actin-binding residues on ADF/cofilin are not well conserved (red bars, Figure 1A ). Therefore, the cellular functions of GMF may have diverged from those of ADF/cofilin [6Goroncy A.K. Koshiba S. Tochio N. Tomizawa T. Sato M. Inoue M. Watanabe S. Hayashizaki Y. Tanaka A. Kigawa T. Yokoyama S. NMR solution structures of actin depolymerizing factor homology domains.Protein Sci. 2009; 18: 2384-2392Crossref PubMed Scopus (34) Google Scholar]. To study the function of S. cerevisiae GMF (YDR063W; also called Aim7, or altered inheritance rate of mitochondria [7Hess D.C. Myers C.L. Huttenhower C. Hibbs M.A. Hayes A.P. Paw J. Clore J.J. Mendoza R.M. Luis B.S. Nislow C. et al.Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis.PLoS Genet. 2009; 5: e1000407Crossref PubMed Scopus (107) Google Scholar]), we first constructed an internal fusion of GFP to GMF, which was functional (see below). GMF-GFP localized to the cytoplasm and cortical puncta. Localization of GMF-GFP in a strain expressing integrated Abp1-RFP, a marker for cortical actin patches [8Kaksonen M. Sun Y. Drubin D.G. A pathway for association of receptors, adaptors, and actin during endocytic internalization.Cell. 2003; 115: 475-487Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar], revealed that most GMF-GFP puncta correspond to actin patches (Figure 1B). However, not all Abp1-RFP patches were labeled with GMF-GFP, suggesting that GMF-GFP may only be present on actin patches for a portion of their lifetime. A similar observation has been made for Cof1-GFP [9Okreglak V. Drubin D.G. Cofilin recruitment and function during actin-mediated endocytosis dictated by actin nucleotide state.J. Cell Biol. 2007; 178: 1251-1264Crossref PubMed Scopus (88) Google Scholar]. Thus, GMF, like Cof1, may be a late-arriving component of actin patches. Next, we made a deletion of the GMF1/AIM7 gene. gmf1Δ (aim7Δ) caused no overt defects in cell growth (Figure 1C) or morphology (not shown), as previously reported [7Hess D.C. Myers C.L. Huttenhower C. Hibbs M.A. Hayes A.P. Paw J. Clore J.J. Mendoza R.M. Luis B.S. Nislow C. et al.Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis.PLoS Genet. 2009; 5: e1000407Crossref PubMed Scopus (107) Google Scholar]. To explore the relationship between GMF and ADF/cofilin, we crossed gmf1Δ to a partial loss-of-function cofilin allele, cof1-22 [10Lappalainen P. Fedorov E.V. Fedorov A.A. Almo S.C. Drubin D.G. Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis.EMBO J. 1997; 16: 5520-5530Crossref PubMed Scopus (203) Google Scholar]. The gmf1Δ cof1-22 double mutant showed more pronounced defects in cell growth compared to cof1-22 after serial dilution and plating (Figure 1D). The synthetic defects were very evident at 34°C, but also could be observed at 25°C by comparing growth rates of strains in liquid medium (Figure 1E). The GMF-GFP construct used above rescued the synthetic growth defects of the gmf1Δ cof1-22 strain (Figure 1F). We investigated whether GMF, like ADF/cofilin, is able to bind and/or sever filamentous actin (F-actin) in vitro. In contrast to Cof1, purified GMF showed minimal cosedimentation with F-actin, even at the highest concentrations tested (10 and 20 μM), when either rabbit muscle actin or yeast actin was used (Figure 2A ). In addition, we failed to detect a binding interaction between GMF and NBD-labeled actin monomers (not shown). Further, GMF did not affect the kinetics of actin assembly or disassembly (Figures 2B–2D), whereas Cof1 accelerated both processes, attributable to its severing activity [11Gandhi M. Achard V. Blanchoin L. Goode B.L. Coronin switches roles in actin disassembly depending on the nucleotide state of actin.Mol. Cell. 2009; 34: 364-374Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar]. GMF also did not enhance actin disassembly effects of Cof1 (Figure 2C), indicating that the two proteins do not synergize in actin severing. Taken together, the genetic, colocalization, and biochemical data suggest that GMF has evolved a function distinct from ADF/cofilin, but one that nevertheless overlaps physiologically with that of ADF/cofilin. To better understand GMF cellular function, we isolated from crude cell extracts proteins that bound to GST-GMF beads but not control GST beads (Figure 2E). Excised gel bands were identified by mass spectrometry as actin, Arp2/3 complex subunits, and the Arp2/3-interacting protein coronin/Crn1 [12Humphries C.L. Balcer H.I. D'Agostino J.L. Winsor B. Drubin D.G. Barnes G. Andrews B.J. Goode B.L. Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin.J. Cell Biol. 2002; 159: 993-1004Crossref PubMed Scopus (146) Google Scholar]. These results suggested an interaction (either direct or indirect) between GMF and Arp2/3 complex, consistent with previous coimmunoprecipitation results in mammalian cells and two-hybrid studies in yeast [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar, 13Uetz P. Hughes R.E. Systematic and large-scale two-hybrid screens.Curr. Opin. Microbiol. 2000; 3: 303-308Crossref PubMed Scopus (100) Google Scholar, 14Yu H. Braun P. Yildirim M.A. Lemmens I. Venkatesan K. Sahalie J. Hirozane-Kishikawa T. Gebreab F. Li N. Simonis N. et al.High-quality binary protein interaction map of the yeast interactome network.Science. 2008; 322: 104-110Crossref PubMed Scopus (1033) Google Scholar, 15Tarassov K. Messier V. Landry C.R. Radinovic S. Serna Molina M.M. Shames I. Malitskaya Y. Vogel J. Bussey H. Michnick S.W. An in vivo map of the yeast protein interactome.Science. 2008; 320: 1465-1470Crossref PubMed Scopus (547) Google Scholar]. Binding assays using purified proteins demonstrated that soluble GMF binds Arp2/3 complex immobilized on beads (Figure 2F), indicating a direct interaction. To study the potential effects of GMF on Arp2/3 complex activity, we first compared Arp2/3 complex-mediated actin assembly kinetics at different concentrations of GMF (Figure 3A ). GMF inhibited Arp2/3 complex-induced actin nucleation in a concentration-dependent manner (Figure 3B) but had no significant effect on actin assembly kinetics in the absence of Arp2/3 complex (Figure 3A and Figure 2B). Half-maximal inhibition of 20 nM Arp2/3 complex was observed at approximately 250 nM GMF, suggesting a submicromolar dissociation constant. To further investigate these effects, we used time-resolved total internal reflection fluorescence (TIRF) microscopy to image nucleation and growth of branched actin filament networks formed by 20 nM Arp2/3 complex (activated by 300 nM VCA) in the presence and absence of 1 μM GMF (Figure 3C). Imaging network growth allowed us to unambiguously identify the mother and daughter filaments at each node. Consistent with the bulk kinetic analysis, GMF caused a marked decrease in rate of daughter nucleation events per unit length of filament (Figure 3D) but no significant change in elongation rate (Figure 3E). Together, these data suggest that binding of GMF to Arp2/3 complex suppresses nucleation of daughter filament assembly. The TIRF experiments were performed under conditions in which actin filaments were not attached to the surface of the microscope slide but could diffuse in two dimensions within the image plane [16Uyeda T.Q. Kron S.J. Spudich J.A. Myosin step size. Estimation from slow sliding movement of actin over low densities of heavy meromyosin.J. Mol. Biol. 1990; 214: 699-710Crossref PubMed Scopus (354) Google Scholar]. In the absence of GMF, branches were stable; detachment was never seen in time-lapse observations of 57 individual branches (lasting on average 10 min from the time each daughter filament began to elongate). However, the addition of low concentrations of GMF (10 nM) induced numerous branch detachments within the first few minutes after daughter nucleation (Figure 4A ). This effect was specific to branch points, because GMF did not induce detectable severing at other locations on filaments. The debranching activity of GMF was potent; it was observed at 10 nM GMF, a concentration below the effective range for inhibition of nucleation and that caused no overt change in rate of branch nucleation or elongation (Figure 3 and data not shown). At this concentration, GMF reduced the characteristic lifetime of branches in 75% of the population to 190 ± 70 s (Figure 4B). The remaining 25% of daughter filaments appeared as a stable fraction unaffected by GMF. This fraction most likely did not debranch simply because insufficient GMF was present in the reaction; much of the GMF present may have been sequestered by Arp2/3 complex, which was in molar excess (20 nM Arp2/3 complex versus 10 nM GMF). At higher GMF concentrations, close to 100% of daughter filaments debranched, and the lifetime was further reduced (120 ± 20 s at 50 nM GMF; 90 ± 20 s at 200 nM GMF). In reactions without GMF, the lifetime was estimated to be longer than 1 hr (see Supplemental Experimental Procedures). The observed debranching kinetics are consistent with a simple mechanism in which GMF binding to Arp2/3 complexes at branch sites induces daughter filament dissociation, and most GMF molecules remain associated with Arp2/3 complex for the duration of the experiment rather than being recycled and made available for subsequent debranching events. Recently, it was shown that ADF/cofilin not only severs F-actin but also stimulates filament debranching by binding to F-actin and promoting dissociation of Arp2/3 complex [4Chan C. Beltzner C.C. Pollard T.D. Cofilin dissociates Arp2/3 complex and branches from actin filaments.Curr. Biol. 2009; 19: 537-545Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. To better understand the respective roles of GMF and ADF/cofilin in remodeling actin networks, we directly compared their effects by TIRF microscopy. In a microscope flow chamber, actin was assembled for 10–15 min with Arp2/3 complex and VCA. Then, buffer containing 50 nM GMF and/or 50 nM Cof1 (without G-actin, Arp2/3 complex, or VCA) was introduced, and filaments were observed for several minutes. Under these conditions, there was no new filament assembly and we could monitor the products of GMF- and Cof1-induced disassembly even though filaments were not tethered to the chamber surface. Exposing branched filaments to GMF produced long, mostly unbranched filaments, whereas addition of Cof1 yielded numerous, very short filaments (Figure 4C). The fragmentation by Cof1 was extensive (Figure 4C) and rapid (data not shown), such that we were unable to assess whether or not some of the filaments were derived from debranching events rather than general severing of F-actin. Nonetheless, we observed that some of the short fragments produced by Cof1 remained branched (Figure 4C). When branched networks were treated with GMF and Cof1 together (50 nM each), the resulting filaments were short and largely unbranched, as expected from the individual effects of both proteins. From these data, it is clear that GMF and Cof1 have strikingly different effects in the remodeling and disassembly of branched filaments, consistent with major differences in their mechanisms (see below). Further analysis is required to learn whether and how the separate effects of GMF and Cof1 might be coordinated to remodel actin networks. The results presented here may help to explain how cellular actin networks are rapidly remodeled during cell motility, endocytosis, cytokinesis, and intracellular transport. Arp2/3 complex binds to sides of preexisting filaments and nucleates formation of daughter filaments at 70° angles, producing branched “dendritic” arrays [17Mullins R.D. Heuser J.A. Pollard T.D. The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments.Proc. Natl. Acad. Sci. USA. 1998; 95: 6181-6186Crossref PubMed Scopus (968) Google Scholar, 18Blanchoin L. Amann K.J. Higgs H.N. Marchand J.B. Kaiser D.A. Pollard T.D. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.Nature. 2000; 404: 1007-1011Crossref PubMed Scopus (420) Google Scholar, 19Amann K.J. Pollard T.D. Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2001; 98: 15009-15013Crossref PubMed Scopus (200) Google Scholar]. These activities play an important role in driving lamellipodial protrusion, endocytic vesicle internalization, and intracellular transport [20Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3105) Google Scholar]. Ultrastructural analyses indicate that Arp2/3-nucleated actin networks at these locations consist of branched filaments [21Svitkina T.M. Borisy G.G. Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.J. Cell Biol. 1999; 145: 1009-1026Crossref PubMed Scopus (864) Google Scholar, 22Taunton J. Rowning B.A. Coughlin M.L. Wu M. Moon R.T. Mitchison T.J. Larabell C.A. Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP.J. Cell Biol. 2000; 148: 519-530Crossref PubMed Scopus (334) Google Scholar, 23Cameron L.A. Svitkina T.M. Vignjevic D. Theriot J.A. Borisy G.G. Dendritic organization of actin comet tails.Curr. Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar], although this remains disputed at the lamellipodial leading edge [24Small J.V. Auinger S. Nemethova M. Koestler S. Goldie K.N. Hoenger A. Resch G.P. Unravelling the structure of the lamellipodium.J. Microsc. 2008; 231: 479-485Crossref PubMed Scopus (50) Google Scholar]. It is not yet understood how branched networks nucleated by Arp2/3 are rapidly disassembled in vivo. Branched filaments assembled by Arp2/3 in vitro persist for tens of minutes ([19Amann K.J. Pollard T.D. Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2001; 98: 15009-15013Crossref PubMed Scopus (200) Google Scholar, 25Mahaffy R.E. Pollard T.D. Kinetics of the formation and dissociation of actin filament branches mediated by Arp2/3 complex.Biophys. J. 2006; 91: 3519-3528Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar] and this study), but in vivo they are dismantled within a few seconds at the leading edge and endocytic loci [20Pollard T.D. Borisy G.G. Cellular motility driven by assembly and disassembly of actin filaments.Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3105) Google Scholar, 26Smith M.G. Swamy S.R. Pon L.A. The life cycle of actin patches in mating yeast.J. Cell Sci. 2001; 114: 1505-1513PubMed Google Scholar]. This suggests that cells may require additional factors to disassemble branched filaments. ADF/cofilin is one factor that stimulates debranching, by binding to F-actin and promoting dissociation of Arp2/3 complex, possibly through propagation of conformational changes in F-actin [4Chan C. Beltzner C.C. Pollard T.D. Cofilin dissociates Arp2/3 complex and branches from actin filaments.Curr. Biol. 2009; 19: 537-545Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. Here we have identified a new role for GMF as a factor that debranches filaments. Although GMF is a structural cousin of ADF/cofilin, it has a distinct mechanism. GMF binds directly to Arp2/3 complex, not actin, and “prunes” daughter filaments at the branch points without severing elsewhere. This produces long unbranched filaments. GMF also inhibits Arp2/3 nucleation of daughter filaments. Both activities, debranching and inhibition of nucleation, may contribute to remodeling the architecture of dendritic actin networks into linear filaments. In contrast, the dual function of ADF/cofilin to sever and debranch filaments leads to fragmentation of branched networks into very short filaments, which can promote their net disassembly, or in other contexts amplify barbed ends to promote new assembly [27Chan A.Y. Bailly M. Zebda N. Segall J.E. Condeelis J.S. Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion.J. Cell Biol. 2000; 148: 531-542Crossref PubMed Scopus (207) Google Scholar]. These observations highlight the mechanistic distinctions between GMF and ADF/cofilin, as well as the targeted specificity of GMF. Interestingly, GMF activities appear to be related to those of coronin. Coronin binds to Arp2/3 complex with high affinity and directly inhibits nucleation [12Humphries C.L. Balcer H.I. D'Agostino J.L. Winsor B. Drubin D.G. Barnes G. Andrews B.J. Goode B.L. Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin.J. Cell Biol. 2002; 159: 993-1004Crossref PubMed Scopus (146) Google Scholar], and more recently it was reported that coronin promotes debranching [3Cai L. Makhov A.M. Schafer D.A. Bear J.E. Coronin 1B antagonizes cortactin and remodels Arp2/3-containing actin branches in lamellipodia.Cell. 2008; 134: 828-842Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar]. Given that we isolated coronin and Arp2/3 complex on GMF affinity columns, this raises the intriguing possibility that GMF and coronin work together in regulating Arp2/3 complex. Based on the activities, localization, and genetic interactions of GMF, we conclude that it may have two different roles in vivo. In some cellular contexts, GMF and ADF/cofilin may work in concert to promote disassembly and turnover of branched actin structures (Figure 4D; coordinated disassembly pathway). In other contexts, GMF may act independently of cofilin to remodel branched networks, by pruning branches and inhibiting nucleation, producing long filaments (Figure 4D; remodeling pathway). Our data also raise many new questions. First, how do low concentrations of GMF dismantle existing branches whereas only higher concentrations prevent nucleation of new branches? The simplest hypothesis is that both activities share the same underlying biochemical mechanism, in which GMF alters Arp2/3 conformation to antagonize its binding to mother filaments. Saturation of Arp2/3 with GMF may be required to effectively block nucleation, whereas only transient binding of GMF may be needed to induce detachment of a branch. Second, did ADF/cofilin and GMF diverge evolutionarily from a common ancestor to interact with actin and actin-related proteins (Arp2 and/or Arp3), respectively? Third, do the activities of S. cerevisiae GMF extend to mammalian homologs? Mammals have two GMF genes, GMF-β and GMF-γ, which together are widely expressed [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar, 28Zaheer A. Fink B.D. Lim R. Expression of glia maturation factor beta mRNA and protein in rat organs and cells.J. Neurochem. 1993; 60: 914-920Crossref PubMed Scopus (64) Google Scholar, 29Walker M.G. Gene expression versus sequence for predicting function: Glia Maturation Factor gamma is not a glia maturation factor.Genomics Proteomics Bioinformatics. 2003; 1: 52-57PubMed Google Scholar]. In addition, GMF-γ has been localized to the leading edge of mammalian cells and coimmunoprecipitates with Arp2/3 complex [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar]. Finally, what mechanisms control the timing of GMF interactions with Arp2/3 complex? This may involve ATP hydrolysis or phosphate release on Arp2 and/or Arp3 [30Martin A.C. Welch M.D. Drubin D.G. Arp2/3 ATP hydrolysis-catalysed branch dissociation is critical for endocytic force generation.Nat. Cell Biol. 2006; 8: 826-833Crossref PubMed Scopus (61) Google Scholar, 31Le Clainche C. Pantaloni D. Carlier M.F. ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays.Proc. Natl. Acad. Sci. USA. 2003; 100: 6337-6342Crossref PubMed Scopus (65) Google Scholar] or in actin subunits in the mother or daughter filaments proximal to branch junctions. There is also the possibility that GMF activity, like that of ADF/cofilin, is controlled by phosphorylation, which has been suggested for mammalian GMF-γ [5Ikeda K. Kundu R.K. Ikeda S. Kobara M. Matsubara H. Quertermous T. Glia maturation factor-gamma is preferentially expressed in microvascular endothelial and inflammatory cells and modulates actin cytoskeleton reorganization.Circ. Res. 2006; 99: 424-433Crossref PubMed Scopus (49) Google Scholar]. For details, see Supplemental Experimental Procedures. Rabbit muscle actin was used in all experiments unless yeast actin is indicated. GMF, Cof1, and VCA fragment of Las17/WASp were purified from E. coli. Arp2/3 complex was purified from S. cerevisiae. Assembly and disassembly assays were performed essentially as described [11Gandhi M. Achard V. Blanchoin L. Goode B.L. Coronin switches roles in actin disassembly depending on the nucleotide state of actin.Mol. Cell. 2009; 34: 364-374Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar] with gel-filtered actin monomers (5% pyrene-labeled) for assembly assays and preassembled F-actin (10% pyrene-labeled) for disassembly assays. In disassembly assays, preassembled F-actin (2 μM) was incubated with GMF and/or Cof1 for 250 s, and then 4 μM Vitamin D-binding protein (human plasma Gc-globulin, Sigma-Aldrich, St. Louis, MO) was added to initiate disassembly. A low copy (CEN) plasmid expressing GMF-GFP was transformed into yeast strain BGY3091, which has an integrated copy of Abp1-RFP. Transformed cells were grown in selective media at 25°C to log phase, then imaged on a Zeiss E600 microscope (Thornwood, NY) equipped with a Hammamatsu Orca ER CCD camera (Bridgewater, NJ) running Openlab software (Improvision Inc., Waltham, MA). RMA was labeled on lysine residues with Alexa-488 NHS (Molecular Probes, Carlsbad, CA), and was mixed at 10% with unlabeled actin (90%), then diluted to 1 μM. Fluorescence emissions were collected on an electron-multiplying charge-coupled device (EMCCD; Andor, South Windsor, CT) for 0.1 s laser exposures at 5 s intervals to generate time-lapse recordings. Analysis of actin elongation and branching rates was performed by tracing filaments as described (see Supplemental Experimental Procedures). Branching rates, kbr, were calculated by counting the number of observed branch nucleation events, Nbr, and dividing by the integral of the total filament length, Ltot, observed over the course of the TIRF recording, according to: kbr=Nbr/∫Ltot(t)dt. Branch nucleation times were calculated by linear extrapolation of daughter filament elongation back to zero length, and branch lifetimes were then calculated as the time from nucleation to the midpoint between the frame in which the branch was last observed and the frame in which the dissociated branch was first observed. We are grateful to G. Rönnholm for assistance with mass spectrometry analysis and to L. Friedman and J. Chung for guidance with TIRF microscopy. This work was supported by grants from the Sigrid Juselius Foundation (to P.L.), from the NIH (GM43369 and GM63007 to J.G., and GM63691 and GM083137 to B.G.), and from NSF (MRSEC to B.G). Download .pdf (.15 MB) Help with pdf files Document S1. Supplemental Experimental Procedures Download .avi (2.3 MB) Help with avi files Movie S1This movie corresponds to the time-lapse series shown in Figure 3C (control); TIRF microscopy of 1 μM actin (10% Alexa-488-labeled) polymerization in the presence of 20 nM Arp2/3 complex and 300 nM VCA. Movie is sped up 50×. Scale bar represents 5 μm. TIRF images (488 nm excitation) were acquired every 5 s. Download .avi (1.98 MB) Help with avi files Movie S2This movie corresponds to the time-lapse series shown in Figure 3C (+ GMF); TIRF microscopy of 1 μM actin (10% Alexa-488-labeled) polymerization in the presence of 20 nM Arp2/3 complex, 300 nM VCA, and 1 μM GMF, showing inhibition of filament branching. Movie is sped up 50×. Scale bar represents 5 μm. TIRF images (488 nm excitation) were acquired every 5 s. Download .avi (.55 MB) Help with avi files Movie S3This movie corresponds to the high-magnification time-lapse series shown in Figure 4A (control); TIRF microscopy of 1 μM actin (10% Alexa-488-labeled) polymerization in the presence of 20 nM Arp2/3 complex and 300 nM VCA, showing representative long-lived branched filaments. Movie is sped up 25×. Scale bar represents 2 μm. TIRF images (488 nm excitation) were acquired every 5 s. Download .avi (3.02 MB) Help with avi files Movie S4This movie corresponds to the time-lapse series used to generate Figure 4A (+ GMF); TIRF microscopy of 1 μM actin (10% Alexa-488-labeled) polymerization in the presence of 20 nM Arp2/3 complex, 300 nM VCA, and 10 nM GMF, showing branched network growth and turnover. Movie is sped up 50×. Scale bar represents 5 μm. TIRF images (488 nm excitation) were acquired every 5 s. Download .avi (.11 MB) Help with avi files Movie S5This movie corresponds to a high-magnification time-lapse series similar to Figure 4A (+ GMF); TIRF microscopy of 1 μM actin (10% Alexa-488-labeled) polymerization in thepresence of 20 nM Arp2/3 complex, 300 nM VCA, and 10 nM GMF, showing representative debranching events. Movie is sped up 25×. Scale bar represents 2 μm. TIRF images (488 nm excitation) were acquired every 5 s. Download .avi (.07 MB) Help with avi files Movie S6This movie corresponds to a high-magnification time-lapse series of a rapid debranching event induced by GMF at 200 nM; TIRF microscopy of 1 μM actin (10% Alexa-488-labeled) polymerization in the presence of 20 nM Arp2/3 complex, 300 nM VCA, and 200 nM GMF. Movie is sped up 25×. Scale bar represents 2 μm. TIRF images (488 nm excitation) were acquired every 5 s." @default.
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• W2023808883 title "GMF Is a Cofilin Homolog that Binds Arp2/3 Complex to Stimulate Filament Debranching and Inhibit Actin Nucleation" @default.
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• W2023808883 cites W1635520329 @default.
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• W2023808883 cites W1973106646 @default.
• W2023808883 cites W1978677736 @default.
• W2023808883 cites W1983813017 @default.
• W2023808883 cites W2006124679 @default.
• W2023808883 cites W2009606782 @default.
• W2023808883 cites W2021087020 @default.
• W2023808883 cites W2033989449 @default.
• W2023808883 cites W2045866553 @default.
• W2023808883 cites W2055188333 @default.
• W2023808883 cites W2056802387 @default.
• W2023808883 cites W2063065938 @default.
• W2023808883 cites W2099573412 @default.
• W2023808883 cites W2104214146 @default.
• W2023808883 cites W2104628504 @default.
• W2023808883 cites W2105789974 @default.
• W2023808883 cites W2108262896 @default.
• W2023808883 cites W2108417233 @default.
• W2023808883 cites W2109248504 @default.
• W2023808883 cites W2112928445 @default.
• W2023808883 cites W2116281786 @default.
• W2023808883 cites W2122782850 @default.
• W2023808883 cites W2126496496 @default.
• W2023808883 cites W2128535856 @default.
• W2023808883 cites W2135342003 @default.
• W2023808883 cites W2167609307 @default.
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