Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.     

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.     

Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.     

Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.     

Close packing of Listeria monocytogenes ActA, a natively unfolded protein, enhances F-actin assembly without dimerization

Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actin-based motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing ( approximately 19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.     

Actin-based motility of pathogens: the Arp2/3 complex is a central player

Bacterial actin-based motility has provided cell biologists with tools that led to the recent discovery that, in many forms of actin-based motilities, a key player is a protein complex named the Arp2/3 complex. The Arp2/3 complex is evolutionally conserved and made up of seven polypeptides involved in both actin filament nucleation and organization. Interestingly, this complex is inactive by itself and recent work has highlighted the fact that its activation is achieved differently in the different types of actin-based motilities, including the well-known examples of Listeria and Shigella motilities. Proteins of the WASP family and small G-proteins are involved in most cases. It is interesting that bacteria bypass or mimic some of the events occurring in eukaryotic systems. The Shigella protein IcsA recruits N-WASP and activates it in a Cdc42-like fashion. This activation leads to Arp2/3 complex recruitment, activation of the complex and ultimately actin polymerization and movement. The Listeria ActA protein activates Arp2/3 directly and, thus, seems to mimic proteins of the WASP family. A breakthrough in the field is the recent reconstitution of the actin-based motilities of Listeria and N-WASP-coated E. coli (IcsA) using a restricted number of purified cellular proteins including F-actin, the Arp2/3 complex, actin depolymerizing factor (ADF or cofilin) and capping protein. The movement was more effective upon addition of profilin, alpha-actinin and VASP (for Listeria). Bacterial actin-based motility is now one of the best-documented examples of the exploitation of mammalian cell machineries by bacterial pathogens.     

Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility

The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes.     

Motility determinants in WASP family proteins

In response to upstream signals, proteins in the Wiskott-Aldrich Syndrome protein (WASP) family regulate actin nucleation via the Arp2/3 complex. Despite intensive study of the function of WASP family proteins in nucleation, it is not yet understood how their distinct structural organization contributes to actin-based motility. Herein, we analyzed the activities of WASP and Scar1 truncation derivatives by using a bead-based motility assay. The minimal region of WASP sufficient to direct movement was the C-terminal WCA fragment, whereas the corresponding region of Scar1 was insufficient. In addition, the proline-rich regions of WASP and Scar1 and the Ena/VASP homology 1 (EVH1) domain of WASP independently enhanced motility rates. The contributions of these regions to motility could not be accounted for by their direct effects on actin nucleation with the Arp2/3 complex, suggesting that they stimulate motility by recruiting additional factors. We have identified profilin as one such factor. WASP- and Scar1-coated bead motility rates were significantly reduced by depletion of profilin and VASP and could be more efficiently rescued by a combination of VASP and wild-type profilin than by VASP and a mutant profilin that cannot bind proline-rich sequences. Moreover, motility of WASP WCA beads was not affected by the depletion or addback of VASP and profilin. Our results suggest that recruitment of factors, including profilin, by the proline-rich regions of WASP and Scar1 and the EVH1 domain of WASP stimulates cellular actin-based motility.     

Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex

ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.     

The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.

Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.     

Capping protein increases the rate of actin-based motility by promoting filament nucleation by the Arp2/3 complex

Capping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of CP and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility.     

Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility

The Listeria monocytogenes surface protein ActA mediates actin-based motility by interacting with a number of host cytoskeletal components, including Ena/VASP family proteins, which in turn interact with actin and the actin-binding protein profilin. We employed a bidirectional genetic approach to study Ena/VASP's contribution to L. monocytogenes movement and pathogenesis. We generated an ActA allelic series within the defined Ena/VASP-binding sites and introduced the resulting mutant L. monocytogenes into cell lines expressing different Ena/VASP derivatives. Our findings indicate that Ena/VASP proteins contribute to the persistence of both speed and directionality of L. monocytogenes movement. In the absence of the Ena/VASP proline-rich central domain, speed consistency decreased by sixfold. In addition, the Ena/VASP F-actin-binding region increased directionality of bacterial movement by fourfold. We further show that both regions of Ena/VASP enhanced L. monocytogenes cell-to-cell spread to a similar degree, although the Ena/VASP F-actin-binding region did so in an ActA-independent manner. Surprisingly, our ActA allelic series enabled us to uncouple L. monocytogenes speed from directionality although both were controlled by Ena/VASP proteins. Lastly, we showed the pathogenic relevance of these findings by the observation that L. monocytogenes lacking ActA Ena/VASP-binding sites were up to 400-fold less virulent during an adaptive immune response.     

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.     

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP''s ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.     

Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.     

Listeria monocytogenes ActA is a key player in evading autophagic recognition

Autophagy is a pivotal bulk degradation system that eliminates undesirable molecules, damaged organelles, and misfolded protein aggregates in response to diverse stimuli, including infection. Autophagy acts to limit intracellular microbial growth but intracellular pathogens have evolved strategies to subvert host autophagic responses for their survival. We found that Listeria monocytogenes ActA, a surface protein required for actin polymerization and actin-based bacterial motility, plays a pivotal role in evading autophagy, but in a manner independent of bacterial motility. We show that L. monocytogenes exploits the biomimetic property of ActA to camouflage itself with host proteins comprised of Ena/VASP and the Arp2/3 complex, thereby escaping recognition by autophagy (Fig. 1).     

Actin filaments align into hollow comets for rapid VASP-mediated propulsion

For cells, the growth of a dense array of branched actin filaments organized by the actin-related proteins 2 and 3 (Arp2/3) complex at the plasma membrane offers an explanation as to how movement is produced, and this arrangement is considered to be optimal for motility. Here, we challenged this assumption by using an in vitro system of polystyrene beads in cell extracts that contained a complex mix of actin polymerization proteins as in vivo. We employed the surface of the bead as a reactor where we mixed two different actin polymerization-activating factors, the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP), to examine their contribution to actin-based movement and filament organization. We varied the coating of the bead surface but left the extracts identical for all assays. We found that the degree of filament alignment in the actin comet tails depended on the surface ratio of VASP to Arp2/3. Alignment of actin filaments parallel to the direction of bead movement in the presence of VASP was accompanied by an abrupt 7-fold increase in velocity that was independent of bead size and by hollowing out of the comets. The actin filament-bundling proteins fimbrin and fascin did not appear to play a role in this transformation. Together with the idea that VASP enhances filament detachment and with the presence of pulling forces at the rear of the bead, a mesoscopic analysis of movement provides a possible explanation for our results.     

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.     

Actin-based motility as a self-organized system: mechanism and reconstitution in vitro

Site-directed actin polymerisation in response to signalling is responsible for the formation of cell protrusions. These elementary 'actin-based motility processes' are involved in cell locomotion, cell metastasis, organ morphogenesis and microbial pathogenesis. We have reconstituted actin-based propulsive movement of particles of various sizes and geometries (rods, microspheres) in a minimum motility medium containing five pure proteins. The ATP-supported treadmilling of actin filaments, regulated by Actin Depolymerizing Factor (ADF/cofilin), profilin and capping proteins provides the thermodynamic basis for sustained actin-based movement. Local activation of Arp2/3 complex at the surface of the particle promotes autocatalytic barbed end branching of filaments, generating a polarized arborescent array. Barbed end growth of branched filaments against the surface generates a propulsive force and is eventually arrested by capping proteins. Understanding the mechanism of actin-based movement requires elucidation of the biochemical properties and mode of action of Arp2/3 complex in filament branching, in particular the role of ATP binding and hydrolysis in Arp2/3, and a physical analysis of the movement of functionalised particles. Because the functionalisation of the particle by an activator of Arp2/3 complex (N-WASP or the Listeria protein ActA) and the concentrations of effectors in the medium are controlled, the reconstituted motility assay allows an analysis of the mechanism of force production at the mesoscopic and molecular levels.     

Follow the monomer

Capping proteins limit actin filament growth, but paradoxically increase actin-based cell motility. This has been attributed to funneling of actin monomers to the filament ends that remain uncapped. Using a reconstituted motility system, Akin and Mullins (2008) now demonstrate that filament capping increases Arp2/3-based nucleation and branching, rather than elevating the rate of filament elongation.