Cellular Microbiology (2000) 2(3), 195205

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

Pascale Cossart Unite des Interactions Bacteries-cellules, Institut Pasteur, Paris 75015, France. Summary 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 actinbased 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, a-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. Introduction A large number of intracellular bacterial pathogens exploit the dynamics of the actin cytoskeleton during infection, but most of them only do so upon entry into mammalian cells. A few of them also use actin for intracellular locomotion (Dramsi and Cossart, 1998). This last category of organisms includes bacteria from three different genera that, after internalization, are able to lyse the phagosomal membrane and reach the cytosol, e.g. Shigella flexneri, Listeria monocytogenes and the closely related species Listeria ivanovii, and Rickettsia conorii and the closely related species Rickettsia rickettsia. All these bacteria can move intra- and intercellularly, resulting in the rapid spread of infection. Intracellular movement is strictly coupled to a polarized actin polymerization process at one pole of the bacterium resulting in the formation of what has been called an actin comet tail or rocket tail. Bacterial movements can reach speeds of mm min21 (e.g. 1090 mm min21 for Listeria). Motility of the Gram-negative bacterium S. flexneri, the first to be discovered, involves an outer membrane protein called VirG or IcsA (Bernardini et al., 1989). The Listeria proteins involved in movement are the surface proteins ActA (for L. monocytogenes) and iActA (for L. ivanovii) which are very similar in structure (Domann et al., 1992; Kocks et al., 1992; Gouin et al., 1995; Kreft et al., 1995). These proteins are anchored in the bacterial membrane, transverse the thick peptidoglycan layer and are exposed to the external milieu. Interestingly, both IcsA and ActA display a polar distribution on the bacterial surface, a feature that appears important for the formation of a polarized actin tail at the bacterial surface in infected cells or in cell extracts. In the case of the strict intracellular bacteria Rickettsia, a gene responsible for actin polymerization has not yet been reported. Actin-based motility is also a property of one enveloped virus, vaccinia virus (Cudmore et al., 1995) in which factors responsible for the phenomenon are beginning to be identified (Frischknecht et al., 1999a). Actin, actin polymerization and actin-based movement
Received 1 February, 2000; revised 12 March, 2000; accepted 28 March, 2000. *For correspondence. E-mail pcossart@pasteur; Tel. (133) 1 45 68 88 41; Fax (133) 1 45 68 87 06. Q 2000 Blackwell Science Ltd

It has been repeatedly stated during the last decade that bacterial motility appears to be a very useful tool for

196 P. Cossart addressing the issue of cellular actin-based movements, such as those of leucocytes during chemotaxis or neurons during outgrowth. As this review will show, bacteria are indeed invaluable tools to elucidate what had long remained a mysterious process. However, one should be aware that, in cells, movements are temporally and spatially controlled by external stimuli transmitted through the plasma membrane. In contrast, bacteria somehow appear as `constitutive moving objects' with no transmission of signals through a membrane. This situation does not imply that bacterial motility is not controlled by a variety of factors, but raises the possibility that more appropriate bacterial models to address the issue of actin polymerization at a cell membrane may be provided by some adherent E. coli strains, in particular EPEC and EHEC strains that induce the formation of actin-rich pedestals on epithelial cells (Sanger et al., 1996; Kalman et al., 1999). Many aspects of the actin polymerization process at the leading edge of a moving cell and at the back end of a bacterium appear similar, with, in particular, the presence of barbed ends at the front of the actin polymerization zone and actin monomers (G-actin) being inserted at the plasma membrane or at the bacterial surface during actin assembly. This led to the key question `what critical factors are required for the actin polymerization process?'. A plethora of actin-binding proteins with different properties had been discovered. Some of these proteins sequester actin monomers and control their availability for the elongation process (e.g. profilin and thymosin b4); some cap the filament barbed ends and regulate/abolish their availability for polymerization (e.g. capping protein); some sever pre-existing filaments inducing the formation of free barbed ends available for actin assembly; and some depolymerize actin and control the dynamics of the process by generating free monomers (e.g. cofilin or ADF). However, nucleators of actin assembly able to create actin filaments (F-actin) with a pointed end and a barbed end from monomeric G-actin had been searched for, but not found. The Arp2/3 complex fulfils such a function and appears as a coordinator of signals to the actin cytoskeleton and an initiator of actin assembly in response to these signals. The next sections will describe the structure of the Arp2/3 complex and our knowledge of how it is activated to become a nucleator of actin assembly. The mechanism for actin assembly utilized by each pathogen will be discussed. Only recent results will be covered in this review (for an earlier review see Dramsi and Cossart, 1998) The Arp2/3 complex The Arp2/3 complex was originally identified using affinity chromatography of Acanthamoeba castellani extracts on a profilin column (Macheski et al., 1994). It has now been isolated from human platelets, bovine brain extracts, Xenopus laevis and Saccharomyces cerevisiae (Welch et al., 1997a; Winter et al., 1997; Machesky and Gould, 1999; Welch, 1999). In mammals, the Arp2/3 complex comprises the two actin-related proteins Arp2 and Arp3 and five additional subunits called p41-Arc, p34-Arc, p21Arc, p-20-Arc and p16-Arc, in apparently stoichiometric amounts. The five Arc proteins have no significant homology to any known proteins. The only striking feature is the presence in p41-Arc of WD repeats, domains thought to mediate protein to protein interactions (Neer et al., 1994). Arp2 and Arp3 are similar to actin in the nucleotide (ATP/ADP) and divalent cation-binding domains, but they are otherwise different from actin and from each other. Both Arp2 and Arp3 fit the structural model of actin with insertions in loops exposed to the solvent. Arp2 has a profilin-binding site that has been confirmed by chemical cross-linking. Both the divergence of the sequences of Arp2 and Arp3 from that of actin in the regions of actin intersubunit contact and the biochemical evidence suggest that neither Arp2 nor Arp3 is able to polymerize actin. Direct contact between Arp2 and Arp3 in the complex in order to mimic an actin dimer able to stimulate the first and limiting step of the actin polymerization process is an appealing possibility (for a review on the structure of the Arp2/3 complex, see Mullins and Pollard, 1999a). The first physiological evidence for a nucleating activity of the Arp2/3 complex was the observation that it could induce actin polymerization at the surface of L. monocytogenes cells (Welch et al., 1997a). A purified fraction from platelet extracts that could induce the formation of an actin cloud when bacteria were mixed with actin was found to contain the Arp2/3 complex. Immunofluorescence studies then revealed that this complex co-localizes not only with the whole Listeria actin tail, but also with the dynamic actin-rich regions of mammalian cells, such as lamellipodia in crawling cells (Machesky et al., 1997; Mullins et al., 1997; Welch et al., 1997b). In vitro, the Arp2/3 complex can enhance, albeit weakly, the rate of actin nucleation, cap the slow-growing (pointed) ends of actin filaments with a high affinity and, interestingly, cross-link actin filaments at a fixed angle (708) (Mullins et al., 1998). Based on these data, the Arp2/ 3 complex was proposed to induce dendritic nucleation of actin filaments at the leading edge of a cell. In this scenario, the Arp2/3 complex nucleates actin filaments which then elongate at their barbed ends. The Arp2/3 complex present at a filament pointed end can also bind to another actin filament, resulting in branch formation. Branched actin filaments have been observed by electron microscopy in the lamellipodia of motile cells and immunoelectron microscopy has shown that the complex
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Actin-based motility is indeed present at filamentfilament junctions (Bailly et al., 1999; Svitkina and Borisy, 1999). Activation of the Arp2/3 complex: role of WASP family proteins and small G proteins A search for factors interacting with the Arp2/3 complex (using the yeast two-hybrid protein interaction assay) revealed that WASP and its relative Scar interact with p21-Arc (Machesky and Insall, 1998). WASP is a protein that is only found in haematopoietic cells (Derry et al., 1994). N-WASP, a closely related protein, is present in most cell types (Miki et al., 1996). Scar was identified in Dictyostelium (Bear et al., 1998). Three isoforms of Scar exist in mammalian cells (one is called Wave). A region present in all members of the family and able to bind to Arp2/3 was identified at the C-terminal part of Scar (Machesky and Insall, 1998). In fact, N-WASP and Bee 1p/Las 17p (the yeast WASP/N-WASP homologue) were later shown to interact with the Arp2/3 complex (Egile et al., 1999; Rohatgi et al., 1999; Winter et al., 1999). The WASP family of proteins including WASP, Scar/Wave, N-WASP and Bee1p/Las17p are modular proteins with domains that interact with various ligands (Bi and Zigmond, 1999; Ramesh et al., 1999). The N-terminal part contains a PH domain (plecktrin homology domain) that is also an EVH1/WH1 (Ena/VASP homology domain 1/WASP homology domain 1), probably able to interact with phospholipids. The presence of such a domain suggests that these proteins could act at the plasma membrane. WASP and N-WASP have a small GTPasebinding domain and bind to Cdc42. All members have a central proline-rich domain that could interact with proteins containing SH3 (Src homology domain 3) domains. This domain is followed by an actin-binding domain called WH2 (or W or V, according to authors). The last part of the protein, called CA (for cofilin homology and acidic) or A according to the authors, is the domain of interaction with the Arp2/3 complex. Overexpression of the CA part of Scar/Wave and WASP disrupt plateletderived growth factor (PDGF)-induced Arp2/3 complex localization and lamellipodia formation (Machesky and Insall, 1998). Several experiments provided evidence that the Arp2/3 complex acts downstream of Cdc42 in a signalling cascade that leads to actin polymerization in extracts of Xenopus laevis eggs, Acanthamoeba or neutrophils (Ma et al., 1998a, 1998b; Huang et al., 1999; Mullins and Pollard, 1999b) These data suggested that WASP proteins could act as adaptors to couple rho family GTPases to the cytoskeleton (Bi and Zigmond, 1999), but how? Once again the bacterial ActA protein played a key role in elucidating this point. Purified ActA and also the N-terminal part of ActA (ActA-N) were shown to stimulate
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the actin nucleation activity of the Arp2/3 complex very efficiently in vitro (Welch et al., 1998). As an extension of the idea that ActA could act as a mammalian protein, ActA was proposed to behave as if it were a member of the WASP family of proteins. This idea was confirmed by the fact that Scar1/Wave, N-WASP, Bee1P/Las17p and WASP all stimulate the nucleating activity of the Arp2/3 complex in vitro (Machesky and Insall, 1998; Egile et al., 1999; Rohatgi et al., 1999; Winter et al., 1999; Yarar et al., 1999). In addition, beads coated with WASP assemble actin at their surface and move in cell extracts in the same way as ActA-coated beads (see below) (Yarar et al., 1999). Thus, the two main challenging questions remaining to be solved were: (1) How are WASP proteins activated? and (2) How do WASP proteins and ActA activate Arp2/3? The way WASP family proteins are activated has been studied in some detail in the case of N-WASP, which appears to be activated by at least Cdc42 and PIP2 (Egile et al., 1999; Rohatgi et al., 1999). Binding to these two compounds would open up the protein and expose the Cterminal region for interaction with the Arp2/3 complex. The mechanism proposed for the activation of the Arp2/ 3 complex is that binding of the complex to the CA region of WASP, N-WASP or an equivalent region in ActA (see below) would bring the Arp2/3 complex next to the actinbinding site WH2/V that, by attracting G-actin, could supply the machinery with polymerization-competent actin monomers. Arp2 and Arp3 might initiate nucleation by forming the initial dimer that is absolutely required for the first nucleation step. It should be noted that, in vitro, the presence of F-actin increases Arp2/3-mediated actin polymerization, suggesting that binding of the complex to F-actin also makes an important contribution in the activation step (Higgs et al., 1999). It is thus possible that the Arp2/3 complex-mediated nucleation process in cells begins from pre-formed filaments.

The Listeria actin-based motility ActA alone can induce actin polymerization and movement The observation that transfection of mammalian cells with the actA gene caused an increase in cellular F-actin content led to the first demonstration that ActA by itself can stimulate actin polymerization (Pistor et al., 1994; Friederich et al., 1995). The ability of ActA to induce not only actin polymerization but also movement was revealed by the fact that the non-motile bacterium L. innocua expressing ActA or Streptococcus pneumoniae asymmetrically coated with ActA could move in cell extracts (Kocks et al., 1995; Smith et al., 1995). The definitive proof that ActA without any other bacterial factor can generate a motile force came from recent studies

198 P. Cossart

Fig. 1. The ActA and IcsA proteins. ActA and IcsA proteins are schematically represented (see text). PS, Signal peptide; MA, membrane anchor.

showing that polystyrene beads coated with purified ActA protein undergo directional movement in an actin-rich cytoplasmic extract (Cameron et al., 1999). Interestingly, small beads (0.5 mM) can easily and spontaneously generate an asymmetry. In contrast, larger beads (up to 2 mM in diameter) can initiate movement only if surface asymmetry is introduced by coating the beads on one hemisphere.

ActA ligands Many actin-binding proteins have been identified in the tails (Dramsi and Cossart, 1998). However, for a long time the only one of these proteins that had been demonstrated to play a role in motility was a-actinin (Dold et al., 1994). Experiments from several groups showed that profilin has an accessory role (Theriot et al., 1994; Marchand et al., 1995; Laurent et al., 1999). The first protein clearly identified as an ActA ligand is VASP (vasodilator stimulated phosphoprotein), a protein found in sites of active actin polymerization and a substrate for c-GMP(PKGs)- and cAMP(PKAs)-dependent kinases (Chakraborty et al., 1995). VASP is the first known proline-rich ligand of profilin. In Listeria-infected cells, VASP is clearly restricted to the bacterial surface at the front of the actin comet tail. It is now well documented that VASP binds to the central proline-rich region of ActA via its EVH1 domain (Niebuhr et al., 1997; Carl et al., 1999). Deletions or mutations in VASP-binding sites of ActA dramatically reduce bacterial speed (Lasa et al., 1995; Smith et al., 1996). VASP also seems to interact with Factin via its C-terminal EVH2 domain, which could help link the bacterium to the tail (Laurent et al., 1999). Interestingly, zyxin, a mammalian docking protein for VASP, shares structural and functional similarities with ActA (Golsteyn et al., 1997). The similarities between ActA and zyxin span the central proline-rich domain and the C-terminal part of ActA. Several approaches have been used to isolate other ActA ligands. The Arp2/3 complex was isolated from human platelet extracts as a `fraction' that could stimulate the formation of an actin cloud around the bacteria (Welch et al., 1997a). This finding initiated the `Arp2/3 boom': at
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The ActA protein ActA is a 610-amino-acid protein that comprises a highly charged N-terminal domain, a central part made of proline-rich repeats and a C-terminal part that anchors the protein in the bacterial membrane (Fig. 1). Genetic analysis has shown that the N-terminal part of ActA is critical for movement (Lasa et al., 1995). Bacteria expressing a chimera made of the N-terminal part of ActA and the v fragment of lacZ can move in cell extracts, showing that the first 233 amino acids of ActA (ActA-N) are sufficient for movement (Lasa et al., 1997). ActA-N is a dimer as shown by the yeast two-hybrid technique and cross-linking experiments directly on the bacteria (Mourrain et al., 1997). The region between amino acids 97 and 127 is critical for dimerization. Fine dissection of ActA-N by deletion analysis pointed to two critical regions that were named T (for tail) and C (for continuity) (Lasa et al., 1997). Region T covers amino acids 116122 KKRRK within the dimerization region and its deletion abrogates tail formation. Deletion of region C (amino acids 2197) leads to discontinuous comet tails. The role of these two regions and of the whole ActA-N is under current investigation.

Actin-based motility the time, the complex was only known for its ability to bind to a profilin column. Interestingly, it is still not clear whether this complex can bind directly to ActA. The use of synthetic peptides corresponding to the regions of ActA important for actin recruitment and propulsion showed that G- and F-actin interact with a region of ActA spanning amino acids 3363 (Lasa et al., 1997). A recent report confirmed the binding of G-actin to purified ActA protein (Cichetti et al., 1999). A novel affinity chromatography approach using whole bacteria was used to isolate proteins that would specifically bind to ActA (David et al., 1998). Proteins known to play a critical role in actin-based movement such as VASP, cofilin and Arp2/3 were detected, as were novel proteins such as Cap Z (which was later shown to be a critical component of the system) and coronin (whose role has yet to be determined). ActA was recently shown to interact with the phospholipid PIP2, but the role of PIP2 is presently unclear (Cichetti et al., 1999; Steffen et al., 1999). One appealing hypothesis is that PIP2 binding unmasks the actin-binding site or other functional sites as in other actin-binding proteins. Finally, using the yeast two-hybrid system, a new ligand of ActA, LaXp180, was identified. LaXp180 binds to recombinant ActA. The site of binding of LaXp180 on ActA and its role in listerial movement are unknown (Pfeuffer et al. 2000). Current model for ActA/Arp2/3 driven motility As already noted, ActA-N was the first polypeptide shown to stimulate actin nucleation in synergy with the Arp2/3 complex (Welch et al., 1997a). The same properties were then demonstrated for Scar, Bee1p, WASP and N-WASP, and their sites of interactions (the CA domain) with the Arp2/3 complex were identified (Egile et al., 1999; Machesky et al., 1999; Rohatgi et al., 1999; Winter et al., 1999; Yarar et al., 1999). Sequence comparisons suggested that the region T in ActA shares some homology with the C region of the WASP family proteins (May et al., 1999). However, a fragment corrresponding to the V region of proteins of the WASP family is not present in ActA. The region spanning amino acids 3363 may play such a role. The A region is also not present. The acidic region present in the N terminal part of ActA could play this role. In summary, the current model suggests that ActA, like the WASP family proteins, recruits and activates the Arp2/3 complex which, in synergy with ActA-N, induces actin polymerization (May et al., 1999). Examination of the actin network formed at the back end of the bacterium clearly shows branched formations, which would be in agreement with the ability of the Arp2/3 complex not only to initiate nucleation process, but also to induce the formation of a dendritic network (Gouin et al., 1999)
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The Shigella actin-based motility The IcsA protein The Shigella IcsA protein, discovered as VirG, is an 1102amino-acid protein that shares no homology with ActA (Lett et al., 1989) (Fig. 1). It can be divided into three domains: a signal peptide (152), an amino terminal domain (53758) and an autotransporter domain (759 1102). The only striking feature of its sequence is the presence of glycine-rich repeats (GRR) in the first part of the protein (103433). Like ActA, IcsA is polarly distributed on the bacterial surface, but its distribution is different with more protein at the poles and less on the sides of the bacteria than in the case of ActA. Shigella cells are unable to move in Xenopus extracts but E. coli K12 strains lacking the outer membrane protease OmpT and expressing IcsA are able to move in the Xenopus extracts and have proved to be very instrumental in analysing IcsA function (Goldberg and Theriot, 1995; Kocks et al., 1995). IcsA ligands Two proteins, vinculin and N-WASP, interact directly with IcsA. Vinculin binds to the region spanning residues 103 508 (Suzuki et al., 1996). It has been reported that infection of cells by Shigella leads to a recruitment of vinculin by IcsA followed by a cleavage of vinculin, which is then able to recruit VASP (Laine et al., 1997). However, the role of vinculin remains controversial as Shigella motility proceeds normally in vinculin-negative cells (Goldberg, 1997). N-WASP is specifically recruited to the pole of intracellular Shigella at the front of the actin tail (Suzuki et al., 1998). This recruitment is due to a specific interaction between the GRR of IcsA and N-WASP. In cos cells expressing an N-WASP variant lacking its C (cofilin) motif, Shigella motility is impaired. Furthermore, depletion of N-WASP completely inhibits comet formation by E. coli (IcsA) in Xenopus extracts (Suzuki et al., 1998; Egile et al., 1999). Current model for IcsA driven actin-based motility IcsA on the bacterial surface has the capacity to recruit NWASP, but this association is not sufficient to induce actin polymerization which also requires the Arp2/3 complex, as shown by the appearance of actin clouds after formation of the tertiary complex IcsA-N-WASP-Arp2/3 (Fig. 2). Interestingly, using purified components it was demonstrated that the ability of IcsA to initiate actin assembly with N-WASP and the Arp2/3 complex is greater than that of Cdc42. These studies have also shown that N-WASP would have, in addition to the

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Fig. 2. The mechanism of IcsA-driven actin-based motility (adapted from Egile et al., 1999). IcsA recruits and opens up N-WASP which becomes able to recruit the Arp2/3 complex which itself becomes activated and able to recruit G-actin and begin to polymerize it. An F-actin binding site in N-WASP allows binding of actin filaments to the bacterial surface. CRIB, Cdc42-Rac interactive binding domain.

capacity to induce actin polymerization via its VCA domain, the ability to bind F-actin via its N-terminal, a property that may be critical for propulsion (Egile et al., 1999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins The respective roles and functions of each partner in actin-based motility has been addressed for a few of the many actin-binding proteins that co-exist in the cells and in comet tails. Carlier and colleagues have now stripped the system down by reconstituting the actin-based motility of Listeria and that of E. coli (IcsA) using pure components in vitro (Loisel et al., 1999). In addition to F-actin, Arp2/3 complex and N-WASP [for E. coli (IcsA)], actin depolymerizing factor (ADF or cofilin) and capping protein were found to be essential for movement. This movement was more effective when profilin, a-actinin and VASP (for Listeria) were added. The authors tested a

wide range of concentrations for each protein and a wide range of combinations of the different components. The system can now be used to establish correlations with in vivo situations. Yet movement in this reconstituted system is slow and it is highly probable that additional regulators remain to be discovered. ActA and IcsA directly or indirectly recruit the Arp2/3 complex to the vicinity of the bacterium. Activation of the Arp2/3 complex initiates actin polymerization through actin monomers that are generated by cofilin, a protein that has affinity for actin-ADP and that stimulates actin depolymerization at pointed ends and, consequently, actin polymerization at barbed ends (Carlier et al., 1997; Rosenblatt et al., 1997). Thus, ADF/cofilin proteins stimulate the dynamics of the process. Their action is enhanced by the presence of profilin which can complex the actin monomer and stimulate a nucleotide exchange reaction to generate polymerization-competent actin monomers (Didry et al., 1999). Capping proteins probably act by what has been called a funneling activity (Carlier and Pantaloni, 1997). In
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Fig. 3. Electron micrographs of actin tails of Listeria (A) and R. conorii (BD) visualized by the technique of myosin S1 decoration (reproduced with permission from J Cell Sci from Gouin et al., 1999).

the situation where barbed ends are capped, actin monomers can only go to the free barbed ends or contribute to the creation of new nucleation sites. Therefore, capping proteins at moderate concentrations might stimulate the process. The situation is not expected to be the same at higher concentrations of capping proteins. This is exactly what is observed experimentally (Loisel et al., 1999). VASP accelerates Listeria movement. Although VASP was originally proposed to recruit profilin which would then recruit actin monomers that could fuel the nucleation/ polymerization complex, an alternative hypothesis proposes that VASP, by binding to F-actin, might help the transient association of actin filaments to the bacteria (Laurent et al., 1999). The same role might be played by the N-terminal part of N-WASP in the case of Shigella. Finally, a-actinin, which cross-links actin filaments, does not influence bacterial movement, but affects the morphology of the tails, suggesting that cross-linking may be important in vivo. As indirectly demonstrated by various techniques, small G proteins do not seem to be involved in the process (Moreau and Way, 1998; Ebel et al., 1999;
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Mounier et al., 1999; V. David and P. Cossart, unpublished results). The actin-based motility of Rickettsia The very closely related species Rickettsia conorii and Rickettsia rickettsii are strict intracellular bacteria for which no genetic tools are available. Recent studies have compared their motilities to that of Listeria and Shigella (Gouin et al., 1999; Heinzen et al., 1999). These bacteria move more slowly, but the actin filaments comprising the actin comet tail are significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100 s vs. 33 s respectively). In addition, in Rickettsia the filaments are much longer than in Listeria or Shigella tails and appear tightly attached to the bacterial surface, at least in the case of R. conorii (Fig. 3). R. rickettsia tails often appear as multiple bundles of actin filaments wrapped around each other in a helical fashion (Heinzen et al., 1999). VASP is present in the tails, as is a-actinin, but it was impossible to detect N-WASP and the Arp2/3 complex, the key players in

202 P. Cossart Listeria and Shigella motility. At present it is not clear whether these proteins were below the limit of detection by the technique used or are really absent. The fact that filaments in the tails are not cross-linked fits well with the idea that Arp2/3 complex is absent. However, this result does not exclude the possibility that the Arp2/3 complex could only be required at the actin nucleation step. It would then detach from the tail and polymerization would progress by a treadmilling mechanism. Such a difference in the behaviour of Rickettsia compared with Listeria and Shigella can be addressed once the bacterial gene responsible for movement is identified. Determination of the sequence of the R. conorii genome is in progress and may help identify candidate genes. Actin-based motility of vaccinia virus The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in cells infected with Listeria and Shigella. Among the six virus-encoded IEV-specific proteins, membrane-anchored protein A36R has been revealed as playing a key role in actin-based motility (Frischknecht et al., 1999a, b). It has a type Ib topology, i.e. a 195-amino-acid cytoplasmic C-terminus tail anchored in the membrane via the N terminus, instead of a type II structure as previously reported (Rottger et al., 1999). This large domain on the surface of the virus membrane becomes tyrosine phosphorylated by Srcfamily kinases on Tyr112 during infection. Tyrosine phosphorylation of A36R results in a direct interaction with the adaptor protein Nck which then recruits N-WASP leading to actin assembly. The scenario that follows is probably similar to that which occurs for Shigella. The observation that vaccinia actin tail formation is dependent on Src-family kinases and tyrosine phosphorylation is reminiscent of signal transduction pathways involved in the control of actin polymerization at the plasma membrane. Concluding remarks Recent months have seen an explosion of new data that indicate a key role for the Arp2/3 complex in actin assembly (Machesky and Gould, 1999; Mullins and Pollard, 1999b; Welch, 1999). This complex is made up of seven polypeptides, the exact roles of which are now being studied. Why so many subunits? Clearly, this complex has to perform many different functions including nucleation, elongation of actin filaments and formation of branched networks. The real breakthrough was the discovery that Arp2/3 can generate branched networks that have been visualized in spectacular electron micrographs of moving cells. This branching explains the burst of actin polymerization that takes place under certain stimuli, such as growth factors, or in highly motile cells. These data have led to the formation of a new principle to explain cell motility, the array treadmilling mechanism (Svitkina and Borisy, 1999). At present, the WASP family proteins are considered to be `the' activators of the Arp2/3 complex, but work in progress in several laboratories (Goode et al., 1999; Jung et al., 1999; Lechler et al., 1999) suggests that other types of activators are also involved. It will be challenging to see how the different activators activate the Arp2/3 complex. While dramatic progress has been made concerning the function of the IcsA protein of Shigella, which appears to be an adaptor that recruits N-WASP, the situation is less clear for the Listeria ActA protein. Genetic dissection of each region of ActA-N should indicate whether the protein really behaves as a WASP family protein. Whether dimer formation plays a role in the control of actin-based motility should also be addressed. Finally, the possible role of PIP2, if any, also remains to be elucidated. The situation in Rickettsia is very intriguing. It is hard to imagine that the Arp2/3 complex is not involved in the movement of this bacterium, even if only transiently. Rickettsia, which are very small bacteria, could transiently use Arp2/3 to initiate actin assembly and induce structures more closely related to filopodia in which Arp2/3 complex is not detected and which do not display branched actin filaments (Castellano et al., 1999). Nevertheless, the presence of long actin filaments in Rickettsia tails has to be explained. Could these filaments be protected from depolymerization by cofilin, which is not detected in the tails? What other component could prevent depolymerization? In any case, Rickettsia tails are unique. The rickettsial protein involved may be very different from IcsA or ActA. The Shigella and Listeria tails that contain the Arp2/3 complex and whose actin filaments appear highly branched appear more closely related to lamellipodia-like structures (Svitkina and Borisy, 1999). In the light of all these new results on actin-based motility, it will be very interesting to see whether invasive bacteria use similar processes of actin polymerization to induce their internalization into mammalian cells.

Acknowledgements
I want to express my special thanks to C. Egile, E. Gouin and R. Jonquieres for help in the figure preparation and to M. Beckerle, V. David, E. Gouin, Tony Pugsley and also the editor D. Sibley for advice on the manuscript. I sincerely acknowledge present and past members of my laboratory, as well as collaborators in France and elsewhere, for their enthusiastic contribution to our study of pathogen actin-based motilities. I also thank L. Blanchoin, M. F. Carlier, D. Drubin, R. Li, L. Macheski, J. B. Marchand, T. Mitchison, D. Pantaloni, T. Pollard and M. Welch for recent stimulating discussions. Work on actin-based motility in my
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Actin-based motility
laboratory received financial support from ARC, Ministere de l'Enseignement Superieur, de la Recherche et de la Technologie and Institut Pasteur.

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