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• W1967975220 abstract "Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals. Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, we establish that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. We propose that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals. Detailed analysis of F-actin dynamics in migrating epithelial cells by quantitative fluorescent speckle microscopy (qFSM) previously revealed two actin modules with distinct dynamic and molecular properties mediating cell protrusion (Ponti et al., 2004Ponti A. Machacek M. Gupton S.L. Waterman-Storer C.M. Danuser G. Two distinct actin networks drive the protrusion of migrating cells.Science. 2004; 305: 1782-1786Crossref PubMed Scopus (601) Google Scholar): the lamellipodium (Lp) and the lamella (Lm). The Lp is an actin network within 1–3 μm of the leading edge characterized by fast retrograde flow and adjacent zones of actin polymerization and depolymerization. The Lm extends from near the leading edge to ∼15 μm toward the cell interior, with slower retrograde flow, and randomly distributed spots of cyclic actin assembly and disassembly (Ponti et al., 2005Ponti A. Matov A. Adams M. Gupton S. Waterman-Storer C.M. Danuser G. Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative Fluorescent Speckle Microscopy.Biophys. J. 2005; 89: 3456-3469Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Molecular components specifically associate with either the Lp or the Lm: the Arp2/3 complex and cofilin, which promote F-actin treadmilling, are concentrated in the Lp (Svitkina and Borisy, 1999Svitkina 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 (905) Google Scholar, Welch et al., 1997Welch M.D. DePace A.H. Verma S. Iwamatsu A. Mitchison T.J. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.J. Cell Biol. 1997; 138: 375-384Crossref PubMed Scopus (406) Google Scholar), while proteins regulating the contractile machinery, myosin II and tropomyosin, localize in the Lm and are excluded from the Lp (Gupton et al., 2005Gupton S.L. Anderson K.L. Kole T.P. Fischer R.S. Ponti A. Hitchcock-DeGregori S.E. Danuser G. Fowler V.M. Wirtz D. Hanein D. Waterman-Storer C.M. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.J. Cell Biol. 2005; 168: 619-631Crossref PubMed Scopus (231) Google Scholar, Ponti et al., 2004Ponti A. Machacek M. Gupton S.L. Waterman-Storer C.M. Danuser G. Two distinct actin networks drive the protrusion of migrating cells.Science. 2004; 305: 1782-1786Crossref PubMed Scopus (601) Google Scholar). Despite the differences between the two F-actin modules, the Lp network may overlap in space with a portion of the Lm (Ponti et al., 2004Ponti A. Machacek M. Gupton S.L. Waterman-Storer C.M. Danuser G. Two distinct actin networks drive the protrusion of migrating cells.Science. 2004; 305: 1782-1786Crossref PubMed Scopus (601) Google Scholar). The mechanisms by which the distinct properties of Lp and Lm are established and maintained, how their interaction affects cell protrusion, and which signals are involved in coregulating the dynamics of the two modules are largely unknown. Resolving these questions is essential to understanding the processes of F-actin-mediated cell protrusion and motility. Due to its concentrated localization at the base of the Lp and its F-actin-severing and -depolymerizing activity (Bamburg, 1999Bamburg J.R. Proteins of the ADF/cofilin family: essential regulators of actin dynamics.Annu. Rev. Cell Dev. Biol. 1999; 15: 185-230Crossref PubMed Scopus (841) Google Scholar), cofilin represents an excellent candidate effector for regulating the interaction between the Lp and Lm networks. Its ability to bind and depolymerize F-actin is inhibited by phosphorylation at serine 3 by the LIM- and TES-family kinases (Toshima et al., 2001Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation.Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (222) Google Scholar, Yang et al., 1998Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.Nature. 1998; 393: 809-812Crossref PubMed Scopus (1067) Google Scholar). LIM kinases (LIMK) are activated by phosphorylation of Thr508/505 (LIMK1/2) through several Rho GTPase-mediated pathways; in particular, Rac/Cdc42 acts through the p21-activated kinase Pak1 (Edwards et al., 1999Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics.Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (845) Google Scholar), and RhoA acts through ROCK (Rho-associated coiled-coil-containing kinase) (Maekawa et al., 1999Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.Science. 1999; 285: 895-898Crossref PubMed Scopus (1284) Google Scholar). Conversely, slingshot (SSH) and chronophin (CIN) have been shown to act as activating phosphatases for cofilin (Huang et al., 2006Huang T.Y. DerMardirossian C. Bokoch G.M. Cofilin phosphatases and regulation of actin dynamics.Curr. Opin. Cell Biol. 2006; 18: 26-31Crossref PubMed Scopus (259) Google Scholar). The consequences of cofilin phosphocycling between active (nonphosphorylated) and inactive (phosphorylated) forms on F-actin dynamics and its downstream effects on cell morphology can be complex. On the one hand, Rho-family GTPases promote F-actin polymerization by activating the Arp2/3 complex (Eden et al., 2002Eden S. Rohatgi R. Podtelejnikov A.V. Mann M. Kirschner M.W. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck.Nature. 2002; 418: 790-793Crossref PubMed Scopus (638) Google Scholar) and/or members of the formin family (Wallar and Alberts, 2003Wallar B.J. Alberts A.S. The formins: active scaffolds that remodel the cytoskeleton.Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) and by inhibiting F-actin depolymerization by cofilin. On the other hand, active cofilin stimulates F-actin severing, thereby initiating the formation of new filament barbed ends that serve as sites for additional F-actin polymerization (Ichetovkin et al., 2002Ichetovkin I. Grant W. Condeelis J. Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex.Curr. Biol. 2002; 12: 79-84Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Active cofilin has also been found to nucleate filaments de novo (Andrianantoandro and Pollard, 2006Andrianantoandro E. Pollard T.D. Mechanism of actin filament turnover by severing and nucleation at different concentrations of ADF/cofilin.Mol. Cell. 2006; 24: 13-23Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), and the depolymerizing function of cofilin is thought to replenish the pool of actin monomers required for further F-actin polymerization (Kiuchi et al., 2007Kiuchi T. Ohashi K. Kurita S. Mizuno K. Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers.J. Cell Biol. 2007; 177: 465-476Crossref PubMed Scopus (143) Google Scholar, Pollard and Borisy, 2003Pollard 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 (3274) Google Scholar). How both cofilin activation and inhibition translate into spatially controlled F-actin dynamics in cells remains unclear. The relationships between cofilin-modulated F-actin activity and the resulting cell morphological responses are similarly complex. Localized activation of cofilin has been shown to promote local edge advancement (Ghosh et al., 2004Ghosh M. Song X. Mouneimne G. Sidani M. Lawrence D.S. Condeelis J.S. Cofilin promotes actin polymerization and defines the direction of cell motility.Science. 2004; 304: 743-746Crossref PubMed Scopus (557) Google Scholar), and pathways have been identified that link cofilin activation to growth factor stimulation in chemotactic protrusion (Chan et al., 2000Chan 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 (210) Google Scholar, Zebda et al., 2000Zebda N. Bernard O. Bailly M. Welti S. Lawrence D.S. Condeelis J.S. Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension.J. Cell Biol. 2000; 151: 1119-1128Crossref PubMed Scopus (169) Google Scholar). This behavior has been explained by cofilin's functions both in generating growing barbed ends and in replenishing the pool of actin monomers. However, the depolymerizing and severing activities of cofilin may also weaken the structure of the F-actin networks, leading to destabilization of the links between Lp, Lm, and/or the cytoplasmic domain of adhesion complexes. Since these links are required to convert the work of growing filaments in the Lp into edge movement, increases in cofilin activity might also be expected to reduce cell protrusivity. In the present study, we analyzed how cofilin function regulates cell protrusion efficiency by differential control of F-actin dynamics in the Lp and the Lm. We perturbed signaling molecules downstream of Rac1, in particular Pak1 and LIMK1, and utilized constructs of constitutively active cofilin to gradually increase cofilin activity from baseline to very high levels. Using immunofluorescence to track changes in molecular components of the Lp and the Lm, combined with qFSM of F-actin dynamics, computational tracking of cell-edge movements, and electron microscopy, our data show that cofilin is a spatial organizer of the Lp and Lm interaction. By this mechanism, it regulates the rates of cellular leading-edge protrusion. We examined the localization of inactive phosphorylated cofilin (pcofilin) to determine the contributions of the Rac1/Pak1/LIMK and RhoA/ROCK/LIMK pathways to the regulation of cofilin activity at the leading edge. These studies utilized PtK1 cells, a marsupial kidney epithelial cell line in which F-actin organization, kinetics, and kinematics have been extensively characterized (Gupton et al., 2005Gupton S.L. Anderson K.L. Kole T.P. Fischer R.S. Ponti A. Hitchcock-DeGregori S.E. Danuser G. Fowler V.M. Wirtz D. Hanein D. Waterman-Storer C.M. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.J. Cell Biol. 2005; 168: 619-631Crossref PubMed Scopus (231) Google Scholar, Gupton and Waterman-Storer, 2006Gupton S.L. Waterman-Storer C.M. Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.Cell. 2006; 125: 1361-1374Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, Ponti et al., 2004Ponti A. Machacek M. Gupton S.L. Waterman-Storer C.M. Danuser G. Two distinct actin networks drive the protrusion of migrating cells.Science. 2004; 305: 1782-1786Crossref PubMed Scopus (601) Google Scholar, Ponti et al., 2005Ponti A. Matov A. Adams M. Gupton S. Waterman-Storer C.M. Danuser G. Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative Fluorescent Speckle Microscopy.Biophys. J. 2005; 89: 3456-3469Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, Wittmann et al., 2003Wittmann T. Bokoch G.M. Waterman-Storer C.M. Regulation of leading edge microtubule and actin dynamics downstream of Rac1.J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (219) Google Scholar). In control cells, pcofilin localized in diffuse punctae throughout protrusions at the cell edge and within the cell body (Figure 1A). Expression of a constitutively active Rac1 mutant (Rac1Q61L, referred to as RacQL) induces the formation of a uniform protrusion all around the unattached cell edge (Wittmann et al., 2003Wittmann T. Bokoch G.M. Waterman-Storer C.M. Regulation of leading edge microtubule and actin dynamics downstream of Rac1.J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (219) Google Scholar) and increases the level of inactive pcofilin in the first 1.5 μm from the leading edge (up 17% compared to control cells) (Figures 1A and 1B). Expression of the Pak1 (auto)inhibitory domain (PID) or a kinase-defective, dominant-negative LIMK1 D460N (LIMK DN) mutant downstream of RacQL decreased pcofilin levels at the leading edge by 33% and 30%, respectively (Figures 1A and 1B). Consistent with these observations, expression of the PID or LIMK DN also modified RacQL-induced actin organization. RacQL-expressing cells present a dense F-actin meshwork in the Lp and dense transverse actin bundles at the base of the Lm (Figure 1A, F-actin, middle panel, arrows). In contrast, an increase in active cofilin downstream of RacQL due to PID or LIMK DN expression induced the formation of a dense F-actin network both in the Lp and in the Lm and of prominent actin bundles in the cell body. Inhibition of ROCK did not significantly decrease leading-edge cofilin phosphorylation in RacQL-expressing cells (12% decrease compared to uninhibited RacQL cells, p = 0.544) (Figures 1A and 1B). However, inhibition of Pak1 or ROCK in PtK1 control cells decreased pcofilin levels at the cell edge by 26% and by 31%, respectively. As a positive control, expression of active Pak1 (Pak H83,86L T423E) or active LIMK (LIMK T508EE) induced an increase of 127% and of 99%, respectively, in pcofilin (Figure 1B; Figure S1, see the Supplemental Data available with this article online). These results indicate that Pak1, but not ROCK, controls cofilin phosphorylation at the leading edge of active Rac1-expressing cells, and that regulation of cofilin via the Pak1/LIMK1 signaling pathway is a required component of Rac1-induced leading-edge actin organization. We used fluorescent speckle microscopy (FSM) to define the effects of Pak1-regulated cofilin activity on actin dynamics at the leading edge (Figures 2A and 2B; Movies S1–S7). In control cells (Movie S1), F-actin underwent a fast retrograde flow in the Lp and a slow retrograde flow in the Lm. Expression of RacQL (Movie S2) did not significantly alter the flow velocity in the Lp (p = 0.992 versus control cells), but it induced a widening of the Lp (Figures 2A and 2B). Inhibition of Pak1 downstream of RacQL (Movie S3) reduced the width of the Lp compared to RacQL alone, and it enhanced the rate of F-actin retrograde flow in the Lp. Pak1 inhibition was also associated with a 2-fold decrease in the rate of F-actin retrograde flow in the Lm, as previously described (Wittmann et al., 2003Wittmann T. Bokoch G.M. Waterman-Storer C.M. Regulation of leading edge microtubule and actin dynamics downstream of Rac1.J. Cell Biol. 2003; 161: 845-851Crossref PubMed Scopus (219) Google Scholar). Importantly, more direct enhancement of cofilin activity downstream of Pak1 by expressing either LIMK DN (Movie S4), an active chronophin phosphatase (CIN) (Movie S5), or a nonphosphorylatable active cofilin (CFL S3A, referred to as CFL SA) (Movie S6) increased the rate of F-actin retrograde flow not only at the leading edge (Figure 2C), but throughout the entire protrusion (Figure 2D). Similar blurring of the gradient in retrograde flow that distinguishes the Lp from the Lm (Figure 2B) was observed with constitutively active cofilin S3A in the absence of a RacQL background (Figures 2B–2D; Movie S7). This further supports the conclusion that the effects on leading-edge dynamics observed are an intrinsic result of cofilin activation. In comparison to control cells, cells with enhanced active cofilin displayed a wider region at the cell edge characterized by rapid actin retrograde flow. This effect could be due to (i) a widening of the Lp, (ii) an inhibition of Lp formation accompanied by an increase of the rate of F-actin retrograde flow in the remaining Lm (Gupton et al., 2005Gupton S.L. Anderson K.L. Kole T.P. Fischer R.S. Ponti A. Hitchcock-DeGregori S.E. Danuser G. Fowler V.M. Wirtz D. Hanein D. Waterman-Storer C.M. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.J. Cell Biol. 2005; 168: 619-631Crossref PubMed Scopus (231) Google Scholar), or (iii) a fusion of the Lp/Lm networks. To distinguish between these possibilities, we used the definition of Lp and Lm as possessing myosin II-insensitive and -sensitive F-actin flows, respectively (Gupton et al., 2005Gupton S.L. Anderson K.L. Kole T.P. Fischer R.S. Ponti A. Hitchcock-DeGregori S.E. Danuser G. Fowler V.M. Wirtz D. Hanein D. Waterman-Storer C.M. Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.J. Cell Biol. 2005; 168: 619-631Crossref PubMed Scopus (231) Google Scholar, Ponti et al., 2004Ponti A. Machacek M. Gupton S.L. Waterman-Storer C.M. Danuser G. Two distinct actin networks drive the protrusion of migrating cells.Science. 2004; 305: 1782-1786Crossref PubMed Scopus (601) Google Scholar). Treatment with blebbistatin, a nonmuscle myosin II ATPase inhibitor (Straight et al., 2003Straight A.F. Cheung A. Limouze J. Chen I. Westwood N.J. Sellers J.R. Mitchison T.J. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor.Science. 2003; 299: 1743-1747Crossref PubMed Scopus (1114) Google Scholar), reduced Lm retrograde flow by 2-fold, but it did not affect Lp flow in control cells (empty vector, EV, Figures 3A–3C; Movie S8). Similar results were obtained in cells expressing RacQL and in cells with enhanced cofilin activity (RacQL and RacQL+CFL SA, Figures 3A–3C; Movies S9 and S10). Spatially resolved maps of F-actin flow speed confirmed that blebbistatin treatment did not affect the characteristic gradient of fast flow at the leading edge to slower flow in the Lm region (Figure 3D). Thus, the broad region of fast F-actin flow in cells with active cofilin is myosin II independent, supporting its definition as Lp. To verify the widening of the Lp in cells with enhanced cofilin activity, we characterized the molecular composition of regions subadjacent to the leading edge. Immunofluorescence localization of high-molecular weight isoforms of tropomyosin established that tropomyosin was reduced near the leading edge of control and active Rac1-expressing cells. Increase of active cofilin levels downstream of Rac1 induced a major depletion of tropomyosin in the entire protrusion (Figure S2A). These observations were confirmed by quantification of the fluorescence intensity ratio of tropomyosin/F-actin from the leading edge into the cell center (Figures S2B–S2D). Tropomyosin was depleted in the first 4 μm adjacent to the cell edge in active Rac1-expressing cells, a distance corresponding to the size of the Lp (Figures 2A and 2B). Cells with enhanced cofilin activity (RacQL+PID, RacQL+LIMK DN, and RacQL+CFL SA) had a 2-fold decreased level of tropomyosin in the first 7.5 μm of the protrusion compared to active Rac1 alone. Similarly, myosin II was absent in the Lp of active Rac1-expressing cells and, interestingly, was depleted much farther from the cell edge in the protrusions of cells with enhanced cofilin activity, as compared to controls (Figures S3A–S3D). These results confirm that activation of cofilin downstream of Rac1 causes the Lp to expand throughout large parts of the protrusion. F-actin flow analysis, coupled with changes in localization of signature molecules, suggested that enhancement of cofilin activity induces the formation of a broad Lp in the protrusion. To verify this hypothesis, we analyzed the spatial organization of F-actin assembly/disassembly rates in these cells (Figure 4A). Actin turnover maps indicated that control cells had a ∼2 μm wide band of strong polymerization along the leading edge (red punctae) juxtaposed to a similarly narrow band of depolymerization (green punctae). Together, these reflect the spatial organization of assembly and disassembly in a treadmilling Lp. The Lm is represented by a region with random foci of weaker polymerization/depolymerization (Figure 4A). Expression of active Rac1 induced actin polymerization in a narrower (∼1 μm), yet more homogeneous, band along the leading edge. Increases of active cofilin markedly modified RacQL-induced F-actin kinetics: expression of PID, LIMK DN, CIN, or CFL SA all induced polymerization extending from the leading edge to deeper within the protrusion (Figure 4A). Behind the wider polymerization band, cells expressing PID still presented a region characteristic of Lm turnover. However, in cells expressing kinase-defective LIMK, CIN, or active CFL, no characteristic pattern of the Lm was observed, and, instead, the region mainly displayed depolymerization events. Again, a similar phenotype was obtained when CFL SA alone was expressed in the absence of active RacQL (Figure 4A). Since cofilin activities include severing/depolymerization (Carlier et al., 1997Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (830) Google Scholar) as well as indirect promotion of barbed end formation and polymerization of F-actin (Condeelis, 2001Condeelis J. How is actin polymerization nucleated in vivo?.Trends Cell Biol. 2001; 11: 288-293Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, Ghosh et al., 2004Ghosh M. Song X. Mouneimne G. Sidani M. Lawrence D.S. Condeelis J.S. Cofilin promotes actin polymerization and defines the direction of cell motility.Science. 2004; 304: 743-746Crossref PubMed Scopus (557) Google Scholar), and may even directly mediate de novo nucleation of filaments (Andrianantoandro and Pollard, 2006Andrianantoandro E. Pollard T.D. Mechanism of actin filament turnover by severing and nucleation at different concentrations of ADF/cofilin.Mol. Cell. 2006; 24: 13-23Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), we further analyzed the localization and density of polymerization-competent free barbed filament ends. In control cells, free barbed ends were distributed in a narrow rim along the leading edge and at the end of F-actin bundles inside the protrusion (Figure 4B). Expression of active Rac1 increased the concentration of free barbed ends that homogeneously localized along the leading edge, in agreement with the narrow band of polymerization measured by qFSM (Figure 4B). In contrast, free barbed filament ends spread widely from the leading edge inside the protrusion of cells with enhanced cofilin activity (Figure 4B: see RacQL+PID, RacQL+LIMK DN, and RacQL+CFL SA). Quantitation of the barbed end fluorescence intensity-to-F-actin intensity ratio (Figures 4C–4E) confirmed that whereas polymerization-competent free barbed filament ends localized in a 0.5–1 μm wide band along the leading edge in RacQL-expressing cells (pink), this band widened in the presence of PID (∼1.5 μm wide band, green), LIMK DN (∼2 μm wide band, red), or CFL SA (∼2.5–3 μm wide band, blue). Several studies suggest a synergy between the cofilin and Arp2/3 pathways, whereby the severing activity of cofilin can amplify the branching activity of the Arp2/3 complex (DesMarais et al., 2004DesMarais V. Macaluso F. Condeelis J. Bailly M. Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension.J. Cell Sci. 2004; 117: 3499-3510Crossref PubMed Scopus (112) Google Scholar, Ichetovkin et al., 2002Ichetovkin I. Grant W. Condeelis J. Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex.Curr. Biol. 2002; 12: 79-84Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Thus, we examined whether increased cofilin activity would broaden the area of Arp2/3 localization in the Lp. Arp2/3 is distributed throughout the cell; the highest concentration is within 1–2 μm from the leading edge (Figure 5A). RacQL expression induced spreading of Arp2/3 into the 2–4 μm region corresponding to the widened Lp (Figure 5A) and also increased its apparent density (Figures 5B–5D, pink). Enhancement of active cofilin downstream of Rac1 reduced the width of the band of Arp2/3 localization (Figure 5A: see RacQL+PID, RacQL+LIMK DN, and RacQL+CFL SA). Quantitation of the fluorescence intensity from the leading edge toward the cell center confirmed that Arp2/3 increased in density but remained localized in the first ∼1–1.5 μm adjacent to the leading edge in cells with enhanced cofilin activity, similar to its localization in control cells (Figures 5B–5D). Thus, enhancement of cofilin activity downstream of Rac1 increases the width of the actin treadmilling array and the density of polymerization-competent filament barbed ends independently of the localization of Arp2/3. To evaluate the role of cofilin in cell protrusion, we investigated the effect of active cofilin on leading-edge dynamics. In control cells, protrusion events propagate as transverse waves of positive velocities along the cell edge (visible in kymographs as diagonal, red stripes) that are intercepted by retraction events (visible as diagonal, blue stripes; Figure 6A; wave pattern highlighted by white lines). In cells expressing RacQL, protrusion of the entire cell edge alternates with retraction of the entire cell edge (visible in kymographs as vertical, red and blue stripes; Figure 6A, dashed, black lines). In cells expressing RacQL+LIMK DN, RacQL+CIN, RacQL+CFL SA, or CFL SA alone (Figure 6A), such patterns of coordinated edge movement are dramatically reduced, suggesting that increased active cofilin disrupts the spatiotemporal coordination of leading-edge movements. We calculated the average net protrusion velocity over multiple cycles to examine how the loss of coordinated movement affected productive edge advancement (Figure 6B, blue bars; the red line indicates increasing active cofilin levels, see Experimental Procedures). Only control cells advanced with a net velocity significantly different from 0 (p = 0.003), compared to cells expressing RacQL, RacQL+LIMK DN, RacQL+CIN, RacQL+CFL SA, or CFL SA alone (p > 0.12 for all conditions). In contrast, the average instantaneous edge velocity was significantly different from 0 for all conditions (Figure 6B, purple bars). Thus, the rate of productive advancement of the leading edge depends primarily on how much time the cell edge spends in a protruding or retracting state, and only secondarily on how fast the edge moves in absolute terms. To test this conclusion further, we defined protrusion efficiency as the ratio between the distances the edge travels in the protruding and the retracting states (Figure 6C). A ratio equal to 1 indicates that protrusion and retraction events cancel one another out, resulting in retention of a constant average position of the cell edge, while a ratio greater than 1 indicates net advancement of the entire leading edge. With the exception of the ratio for control cells, none of the protrusion efficiency scores in the other" @default.
• W1967975220 created "2016-06-24" @default.
• W1967975220 creator A5005929667 @default.
• W1967975220 creator A5026554721 @default.
• W1967975220 creator A5033451222 @default.
• W1967975220 creator A5037958980 @default.
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• W1967975220 date "2007-11-01" @default.
• W1967975220 modified "2023-10-14" @default.
• W1967975220 title "Cofilin Activity Downstream of Pak1 Regulates Cell Protrusion Efficiency by Organizing Lamellipodium and Lamella Actin Networks" @default.
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