A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility

Spotted fever group Rickettsia are obligate intracellular pathogens that exploit the host cell actin cytoskeleton to promote motility and cell-to-cell spread. Although other pathogens such as Listeria monocytogenes use an Arp2/3 complex-dependent nucleation mechanism to generate comet tails consisting of Y-branched filament arrays, Rickettsia polymerize tails consisting of unbranched filaments by a previously unknown mechanism. We identified genes in several Rickettsia species encoding proteins (termed RickA) with similarity to the WASP family of Arp2/3-complex activators. Rickettsia rickettsii RickA activated both the nucleation and Y-branching activities of the Arp2/3 complex like other WASP-family proteins, and was sufficient to direct the motility of microscopic beads in cell extracts. Actin tails generated by RickA-coated beads consisted of Y-branched filament networks. These data suggest that Rickettsia use an Arp2/3 complex-dependent actin-nucleation mechanism similar to that of other pathogens. We propose that additional Rickettsia or host factors reorganize the Y-branched networks into parallel arrays in a manner similar to a recently proposed model of filopodia formation.     

Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility

Diverse intracellular pathogens subvert the host actin-polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive 'comet tails' that consist of long, unbranched actin filaments. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks. However, a second bacterial gene, sca2, was recently implicated in actin-tail formation by R. rickettsii. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators.     

Arp2/3 is a negative regulator of growth cone translocation

Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.     

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.     

Actin filament attachments for sustained motility in vitro are maintained by filament bundling

We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+) generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+) abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+), Lys-Lys(2+), or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+) buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.     

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.     

Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.     

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.     

Formation of filopodia-like bundles in vitro from a dendritic network

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.     

The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins

The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.     

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.     

Integration of linear and dendritic actin nucleation in Nck-induced actin comets

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails—dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.     

Cortactin promotes cell motility by enhancing lamellipodial persistence

BACKGROUND: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS: Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS: Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility.     

Interaction of SPIN90 with the Arp2/3 complex mediates lamellipodia and actin comet tail formation

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.     

Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.     

Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis

Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.     

Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.