Abstract: The actin cytoskeleton of a eukaryotic cell is critical for many functions, including cell locomotion, cytokinesis, intracellular transport, establishment of cell polarity, and the elaboration of cell surface projections that increase cell surface area and sense the environment. Arrays of actin filaments must be assembled within cells in a fashion that is both spatially and temporally controlled in order to contribute to such a diversity of cellular processes. Cells face a number of challenges when it comes to actin. Actin filaments are structurally and functionally polar polymers that assemble from monomers that come together in a head to tail fashion. The intrinsic polarity of an actin filament can be visualized by decoration of the filament with myosin fragments that coat it to generate an arrowhead appearance. One end of the filament, referred to as the barbed or "plus" end is the preferred site for monomer addition; the other end, referred to as the "minus" end, is the slow end with respect to growth. Actin polymerization occurs in two phases. The rate-limiting step in actin filament formation involves the generation of an assembly-competent actin nucleus such as an actin trimer by de novo assembly or a free actin filament end by uncapping or severing. When an actin nucleus is present and the concentration of monomer is sufficient, spontaneous elongation of a filament occurs. Within cells, monomeric actin is present at concentrations well above what is required for rapid polymerization at physiological ionic strength. So, what is it that prevents our cells from filling up with tangled masses of filamentous actin? One way that cells control actin assembly is by sequestering monomeric actin subunits through stoichiometric binding to protein partners such as thymosin β4 and profilin that control the availability of the actin monomers for polymerization. Cells also produce proteins such as CapZ and gelsolin that can cap the fast-growing (barbed) ends of actin filaments and thus control the elongation of preexisting filaments. Control of nucleation and regulation of available actin monomer levels thus provide important mechanisms for regulating the amount of actin polymer inside cells, but they are not alone sufficient to answer the fundamental question of how these actin filaments come to be assembled at appropriate locations within the cell. Simple inspection of cellular architecture makes it clear that cells have exquisite control over where actin filaments will accumulate. In cochlear hair cells, highly ordered apical bundles of actin filaments form the structural backbone of the stereocilia that sense vibration (Figure 1A). In striated muscle, semicrystalline arrays of actin and myosin are constructed to establish the contractile machinery (Figure 1B). Although many cells lack such striking actin filament displays, they nevertheless exhibit a similar capacity to define zones of actin assembly. Processes such as cell migration depend on the ability of cells to fix sites of actin assembly with a high degree of spatial resolution. For example, for vectorial cell locomotion of a fibroblast to occur, actin assembly must be focused at the leading edge (Figure 1C), a broad membrane protrusion that is comprised of a meshwork of actin filaments that are generally oriented with their barbed ends directed toward the cell periphery. The situation for actin contrasts with that of its sister polymer, the microtubule. In animal cells, there is typically a single cellular site called the microtubule organizing center that provides the primary focus for microtubule assembly. An excess of microtubules is assembled with apparent spatial abandon, and only selected microtubules are retained by stabilization, for example by binding to specialized capture sites on mitotic chromosomes. For the actin cytoskeleton, assembly appears to follow site selection. But how are suitable sites for actin assembly specified? One particularly useful model organism for deciphering the mechanism by which spatially controlled actin assembly is accomplished in mammalian cells is the intracellular bacterial pathogen, Listeria (42Tilney L.G Portnoy D.A Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite Listeria monocytogenes.J. Cell Biol. 1989; 109: 1597-1608Crossref PubMed Scopus (910) Google Scholar). Listeria is a Gram-positive food-borne pathogen that can cause life-threatening illnesses such as encephalitis, particularly in immune-compromised individuals. Listeria has the capacity to invade mammalian cells. Once internalized, the bacterium escapes the membrane-bound endosome, replicates, and resides happily within the host cytoplasm. There, the bacterium harnesses the host machinery required for actin assembly and generates an actin "comet" tail (Figure 1D) that propels the bacterium within the host cell. The intracellular motion of Listeria is directly coupled to actin assembly and is independent of myosin function (36Sanger J.M Sanger J.W Southwick F.S Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes.Infect. Immun. 1992; 60: 3609-3619Crossref PubMed Google Scholar; 40Theriot J.A Mitchison T.J Tilney L.G Portnoy D.A The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.Nature. 1992; 357: 257-260Crossref PubMed Scopus (396) Google Scholar). It is this actin-based motility of the bacterium that enables it to enter an adjacent cell via a membrane-bound projection. Because the ability of Listeria to be transferred from one cell to the next, and thus to spread the infection, is absolutely dependent on its ability to build an actin-rich comet tail, microbiologists have sought to identify bacterial genes that are critical for this process. Interestingly, just a single bacterial factor, the ActA protein, has been shown to be required for the ability of Listeria to assemble filamentous actin on its surface within the host cytoplasm (7Domann E Wehland J Rohde M Pistor S Hartl M Goebel W Leimeister-Wächter M Wuenschner M Chakraborty T A novel bacterial gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.EMBO J. 1992; 11: 1981-1990Crossref PubMed Scopus (318) Google Scholar; 17Kocks C Gouin E Tabouret M Berche P Ohayon H Cossart P Listeria monocytogenes-induced actin assembly requires the actA gene product, a surface protein.Cell. 1992; 68: 521-531Abstract Full Text PDF PubMed Scopus (610) Google Scholar). ActA is asymmetrically distributed on the bacterial surface, concentrated on the end of the pathogen that is associated with the actin comet tail. Although the ActA protein is not alone sufficient to stimulate the assembly of biochemically purified actin, a number of lines of evidence suggest that it drives actin assembly within the context of eukaryotic cytoplasm. For example, when ActA bearing a C-terminal CAAX sequence that signals farnesylation and carboxymethylation is targeted to the inner leaflet of the plasma membrane of uninfected mammalian cells, it induces the elaboration of actin-rich cell surface projections (9Friederich E Gouin E Hellio R Kocks C Cossart P Louvard D Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to dissect both actin-based cell morphogenesis and ActA function.EMBO J. 1995; 14: 2731-2744Crossref PubMed Scopus (67) Google Scholar). Likewise, targeting of ActA to the surface of mitochondria stimulates organelle-associated actin assembly (31Pistor S Chakraborty T Niebuhr K Domann E Wehland J The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.EMBO J. 1994; 13: 758-763Crossref PubMed Scopus (148) Google Scholar), and the expression of ActA by a nonpathogenic bacterium is sufficient to promote comet tail formation (18Kocks C Marchand J.B Gouin G d'Hauteville H Sansonetti P.J Carlier M.F Cossart P The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocula and Escherichia coli respectively.Mol. Microbiol. 1995; 18: 413-423Crossref PubMed Scopus (145) Google Scholar). Because of the ability of ActA to interact so productively with host factors involved in actin assembly, it has been speculated that ActA is a molecular mimic of a mammalian protein or proteins that play central roles in the process of actin assembly. Thus, understanding precisely how ActA works is likely to provide substantial insight into how cells define sites of actin assembly. Recently, much progress has been made toward determining the role of the ActA protein and its relationship to the endogenous cellular machinery involved in actin assembly. The functional domains of the ActA protein have been defined by mutagenesis (Figure 2) (19Lasa I David V Gouin E Marchand J.P Cossart P The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator.Mol. Microbiol. 1995; 18: 425-426Crossref PubMed Scopus (93) Google Scholar; 32Pistor S Chakraborty T Walter U Wehland J The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins.Curr. Biol. 1995; 5: 517-525Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; 38Smith G.A Theriot J.A Portnoy D.A The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.J. Cell Biol. 1996; 135: 647-660Crossref PubMed Scopus (182) Google Scholar; 26Mourrain P Lasa I Gautreau A Gouin E Pugsley A Cossart P ActA is a dimer.PNAS. 1997; 94: 10034-10039Crossref PubMed Scopus (31) Google Scholar). The ActA protein, which exists as a dimer, is tethered to the bacterial surface via a C-terminal anchor. As might be predicted for a protein that stimulates the assembly of such glorious actin arrays within cells (Figure 1D), ActA displays several regions that promote actin assembly. An N-terminal ActA domain is absolutely required for the ability of the bacterium to elaborate actin filament arrays on its surface. As will be discussed in greater detail below, this region of ActA has recently been implicated in facilitating actin nucleation, the rate-limiting step in actin assembly. A central proline-rich domain of ActA is also necessary for efficient assembly of the comet tail but is not in itself sufficient to trigger actin assembly. Deletions in the proline region of ActA slow the rate of bacterial motility, a process that is directly coupled to actin polymerization, thus there is a functional link between the proline-rich region and actin assembly. The mechanism by which the proline-rich domain augments net actin polymerization on the bacterial surface is not well understood. Several possibilities remain consistent with experimental observations. For example, this region could act directly or indirectly to enhance the nucleation potential of the N-terminal domain of ActA, could stimulate actin filament elongation by promoting filament uncapping or monomer addition, or could somehow enhance local levels of polymerization-competent monomer. Reconstitution of ActA-dependent actin assembly with purified components will be necessary to distinguish among these (and other) possibilities. It is clear that ActA requires the cooperation of several host factors to stimulate actin polymerization. One interesting view is that the ActA protein represents a hybrid protein that exhibits functions found in multiple eukaryotic proteins; noncovalent association of these cellular proteins would reconstitute full "ActA activity" and provide a cellular machine to regulate actin assembly. Perhaps ancestors of Listeria acquired genes related to those of their early mammalian hosts by nucleic acid transfer. If that were the case, then one would expect to be able to identify relatives of ActA in mammalian cells. Alternatively, it is also possible that Listeria evolved entirely novel strategies for interacting with the host machinery for actin assembly. In either case, identification of the host components that associate with ActA has already provided insight into how cells regulate the polymerization of actin. Reconstitution of actin assembly and Listeria motility in cytoplasmic extracts has led to the identification of host proteins that collaborate with ActA to promote actin assembly in vivo. Using human platelet extracts to support ActA-dependent actin assembly on the surface of Listeria, Welch and colleagues showed that a complex of proteins called the Arp2/3 complex is both necessary and sufficient for ActA's ability to provide a focus for actin assembly (44Welch M.D Iwamatsu A Mitchison T.J Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes.Nature. 1997; 385 (a): 265-269Crossref PubMed Scopus (479) Google Scholar). The Arp2/3 complex is highly conserved from Acanthamoeba to humans (16Kelleher J.F Atkinson S.J Pollard T.D Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (153) Google Scholar; 22Machesky L.M Reeves E Wientjes F Mattheyse F.J Grogan A Totty N.F Burlingame A.L Hsuan J.J Segal A.W Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins.Biochem. J. 1997; 328: 105-112Crossref PubMed Scopus (169) Google Scholar; 45Welch M.D DePace A.H Verma S Iwamatsu A Mitchison T.J The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.J. Cell Biol. 1997; 138 (b): 375-384Crossref PubMed Scopus (382) Google Scholar) and contains seven protein constituents, including two a ctin-r elated p roteins, Arp2 and Arp3. As would be expected for a collection of proteins that play a central role in actin polymerization, the Arp2/3 complex is enriched at sites of actin assembly such as the leading edges of motile cells (16Kelleher J.F Atkinson S.J Pollard T.D Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (153) Google Scholar; 22Machesky L.M Reeves E Wientjes F Mattheyse F.J Grogan A Totty N.F Burlingame A.L Hsuan J.J Segal A.W Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins.Biochem. J. 1997; 328: 105-112Crossref PubMed Scopus (169) Google Scholar; 45Welch M.D DePace A.H Verma S Iwamatsu A Mitchison T.J The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.J. Cell Biol. 1997; 138 (b): 375-384Crossref PubMed Scopus (382) Google Scholar). Arp2 and Arp3 have also been identified in yeast where they have been shown to be localized in actin-rich cortical structures and are required for actin-dependent functions (24McCollum D Feoktistova A Morphew M Balasubramanian M Gould K.L The Schizosaccharomyces pombe actin-related protein, Arp3, is a component of the cortical actin cytoskeleton and interacts with profilin.EMBO J. 1996; 15: 6438-6446Crossref PubMed Scopus (113) Google Scholar; 25Moreau V Madania A Martin R.P Winson B The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton.J. Cell Biol. 1996; 134: 117-132Crossref PubMed Scopus (110) Google Scholar). Because of the presence of two actin-related proteins in the Arp2/3 complex, it was postulated that the Arp2/3 complex might stimulate actin assembly by mimicking the structure of an actin nucleus or free filament end (16Kelleher J.F Atkinson S.J Pollard T.D Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (153) Google Scholar). However, in recent biochemical studies of Acanthamoeba and human Arp2/3, the complex failed to reduce significantly the rate-limiting step for actin assembly (27Mullins R.D Heuser J.A Pollard T.D The interaction of Arp2/3 complex with actin nucleation, high affinity pointed end capping, and formation of branching networks of filaments.PNAS. 1998; 95 (a): 6181-6186Crossref PubMed Scopus (968) Google Scholar; 46Welch M.D Rosenblatt J Skoble J Portnoy D.A Mitchison T.J Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.Science. 1998; 281: 105-108Crossref PubMed Scopus (404) Google Scholar); thus, it does not appear to act as a preformed nucleus for actin assembly, at least not on its own. The fact that the Arp2/3 complex is an inefficient nucleator of actin assembly when it is in a highly purified form provides a potentially powerful mechanism for a cell to control its activity by regulating the availability and/or localization of essential cofactors. In a Listeria-infected cell, the ActA protein on the bacterial surface appears to function as the cofactor that stimulates the nucleating activity of Arp2/3 (46Welch M.D Rosenblatt J Skoble J Portnoy D.A Mitchison T.J Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.Science. 1998; 281: 105-108Crossref PubMed Scopus (404) Google Scholar). Although direct binding of Arp2/3 to ActA has not been demonstrated, it is pleasing that the domain of ActA that was found to synergize with Arp2/3 to nucleate actin assembly in vitro maps to the N terminus of the protein, the same region of ActA that has been shown to be essential for nucleation of actin assembly on the surface of the bacterium within the cytoplasm of a mammalian cell. The precise mechanism by which the Arp2/3 complex stimulates actin assembly is not understood. Although the Arp2/3 complex does not act as a preformed nucleus for polymerization on its own, it may have this property in the presence of key accessory factors like Listeria ActA. Indeed, when ActA and Arp2/3 are mixed together with monomeric actin, the nucleation phase of actin assembly, which is normally slow, is essentially instantaneous (46Welch M.D Rosenblatt J Skoble J Portnoy D.A Mitchison T.J Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.Science. 1998; 281: 105-108Crossref PubMed Scopus (404) Google Scholar). The ability of purified Arp2/3 to bind to the pointed, slow-growing ends of actin filaments has also led to the suggestion that the Arp2/3 complex may facilitate actin assembly by docking and stabilizing an actin nucleus such as a dimer or trimer (27Mullins R.D Heuser J.A Pollard T.D The interaction of Arp2/3 complex with actin nucleation, high affinity pointed end capping, and formation of branching networks of filaments.PNAS. 1998; 95 (a): 6181-6186Crossref PubMed Scopus (968) Google Scholar). Arp2/3-dependent capping of actin filaments may also enhance their stability by slowing disassembly from the pointed ends of the polymers. The ability of the Arp2/3 complex to cap the pointed ends of actin filaments may help to explain why Arp2/3 is found throughout the actin comet tail of a motile Listeria, rather than just at the site of actin growth near the bacterial surface. Many of the actin filaments found in the Listeria comet tail are short relative to the length of the tail. If short actin filaments nucleated by Arp2/3 in close proximity to ActA were subsequently released with an Arp2/3 cap, the Arp2/3 complex might persist in the older portions of the tail as ActA-associated actin assembly progressed near the bacterial surface. In addition to its involvement in the nucleation of actin assembly, the Arp2/3 complex has also been implicated in the organization of actin arrays. In vitro, the Arp2/3 complex promotes the assembly of branched actin filament arrays. It has been suggested that the Arp2/3 complex may support the assembly of a new filament off the side of a preexisting polymer (27Mullins R.D Heuser J.A Pollard T.D The interaction of Arp2/3 complex with actin nucleation, high affinity pointed end capping, and formation of branching networks of filaments.PNAS. 1998; 95 (a): 6181-6186Crossref PubMed Scopus (968) Google Scholar). The morphology of actin filament arrays that are prepared in the presence of Arp2/3 is similar to the filament architecture observed at the leading edge of cells (39Svitkina T.M Verkhovsky A.B McQuade K.M Borisy G.G Analysis of the actin-myosin II system in fish epidermal keratocyctes mechanism of cell body translocation.J. Cell. Biol. 1997; 139: 397-415Crossref PubMed Scopus (532) Google Scholar), an observation that has suggested a role for Arp2/3 proteins in lamellipodial cytoarchitecture. The illustration that Arp2/3 and ActA cooperate to stimulate actin nucleation suggests that one way to control actin assembly would be to control the access of these two components to each other. Localization of the Arp2/3 complex, its cellular activator(s), or both to specific cellular sites is likely to be critical for defining fertile zones for actin assembly in uninfected cells as well. Identification of the endogenous cellular factors that cooperate with the Arp2/3 complex to nucleate actin polymerization will surely provide substantial insight into how actin assembly sites are generated within cells. Examination of the region of ActA involved in acceleration of actin polymerization has also provided insight into the cellular mechanisms required for assembly of actin filaments. The assembly-enhancing activity of ActA has been mapped to a series of short proline-rich repeats characterized by the consensus D/EFPPPP. Mutations that affect these proline repeats result in a reduction in actin polymer on the surface of the bacterium and a reduced rate of motility of the bacterium within the host cytoplasm (19Lasa I David V Gouin E Marchand J.P Cossart P The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator.Mol. Microbiol. 1995; 18: 425-426Crossref PubMed Scopus (93) Google Scholar; 32Pistor S Chakraborty T Walter U Wehland J The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins.Curr. Biol. 1995; 5: 517-525Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar; 38Smith G.A Theriot J.A Portnoy D.A The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.J. Cell Biol. 1996; 135: 647-660Crossref PubMed Scopus (182) Google Scholar). The proline repeats of ActA have been shown to serve as docking sites for host proteins in the Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) family (38Smith G.A Theriot J.A Portnoy D.A The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.J. Cell Biol. 1996; 135: 647-660Crossref PubMed Scopus (182) Google Scholar; 29Niebuhr K Ebel F Frank R Reinhard M Domann E Carl U.D Walter U Gertler F.B Wehland J Chakraborty T A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family.EMBO J. 1997; 16: 5433-5444Crossref PubMed Scopus (325) Google Scholar). To date, Ena/VASP family members are the only known ligands for these bioactive proline-rich sequences, so it is reasonable to imagine that the enhancement of actin assembly by the proline repeats involves Ena/VASP activity. In support of this view, at least one mammalian Ena/VASP family member triggers actin assembly when expressed in mammalian cells (11Gertler F.B Niebuhr K Reinhard M Wehland J Soriano P Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). However, it should be noted that no direct evidence that Ena/VASP proteins are essential for optimal assembly of the Listeria comet tail has yet been presented. A demonstration that cells or extracts lacking Ena/VASP activity show reduced ability to support actin assembly and bacterial motility would confirm directly a role for these proteins in comet tail formation. Nevertheless, given the reasonable hypothesis that Ena/VASP proteins are central to Listeria comet tail assembly, how might they enhance actin polymerization at the bacterial surface? One potential mechanistic link between Ena/VASP proteins and actin polymerization is derived from the ability of these proteins to multimerize and dock profilin, an actin monomer binding protein that stimulates the assembly of actin filaments at their barbed ends (33Reinhard M Giehl C Abel K Haffner C Jarchau T Hoppe V Jockusch B.M Walter U The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.EMBO J. 1995; 14 (a): 1583-1589Crossref PubMed Scopus (408) Google Scholar; 15Kang F Laine R.O Rubb J.R Southwick F.S Purich D.L Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP) implications for actin-based Listeria motility.Biochemistry. 1997; 36: 8384-8392Crossref PubMed Scopus (103) Google Scholar). Localization studies have revealed that both Ena/VASP proteins and profilin are closely associated with the actin assembly-competent end of a Listeria cell (41Theriot J.A Rosenblatt J Portnoy D.A Goldschimdt-Clermont P.J Mitchison T.J Involvement of profilin in the actin-based motility of L. monocytogenes in cells and cell-free extracts.Cell. 1994; 76: 505-517Abstract Full Text PDF PubMed Scopus (241) Google Scholar; 11Gertler F.B Niebuhr K Reinhard M Wehland J Soriano P Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics.Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar; 38Smith G.A Theriot J.A Portnoy D.A The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.J. Cell Biol. 1996; 135: 647-660Crossref PubMed Scopus (182) Google Scholar). It has been suggested that the ActA-dependent recruitment of Ena/VASP proteins and their partners could serve to concentrate actin monomers at the bacterial surface, close to the region where assembly-competent actin nuclei are found. In theory, ActA could serve as a docking site for up to four Ena/VASP family members, since it displays four proline-rich repeats and VASP is a tetramer with numerous potential profilin-binding sites; therefore, if all VASP-binding and profilin-binding sites were fully occupied, a dramatic increase in local profilin–actin levels could be achieved. This view is appealing in its simplicity, but it is not clear how the accumulation of profilin–actin bound to Ena/VASP family members might contribute to enhancing local levels of soluble profilin–actin pools that would be available to participate in actin filament elongation. Moreover, the very role of profilin in the assembly of the Listeria tail remains controversial, since depletion of profilin has not consistently abolished comet tail assembly (41Theriot J.A Rosenblatt J Portnoy D.A Goldschimdt-Clermont P.J Mitchison T.J Involvement of profilin in the actin-based motility of L. monocytogenes in cells and cell-free extracts.Cell. 1994; 76: 505-517Abstract Full Text PDF PubMed Scopus (241) Google Scholar; 23Marchand J.-P Moreau P Paoletti A Cossart P Carlier M.-F Pantaloni D Actin-based movement of Listeria monocytogenes actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface.J. Cell Biol. 1995; 130: 331-343Crossref PubMed Scopus (116) Google Scholar). Clearly, there are some missing links in our understanding of the biological roles of Ena/VASP proteins. Perhaps they act catalytically to enhance the assembly potential of actin–profilin complexes in their vicinity or to transfer actin monomers directly to nearby filament ends. Alternatively, the Ena/VASP–profilin complex could have an impact on Arp2/3's nucleation activity; Acanthamoeba Arp2/3 binds profilin (28Mullins R.D Kelleher J.F Xu J Pollard T.O Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments.Mol. Cell Biol. 1998; 9 (b): 841-852Crossref Scopus (68) Google Scholar), though the biological consequences of this association, if any, have not been elucidated. Two potential counterparts of the proline-rich region of the ActA protein have been identified in metazoans, including mammals. A proline repeat similar to those found in ActA is present in vinculin, a component of adhesion plaques that also binds VASP (4Brindle N.P.J Holt M.R Davies J.E Price C.J Critchley D.R The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin.Biochem. J. 1996; 318: 753-757Crossref PubMed Scopus (157) Google Scholar); however, the ability of vinculin to cooperate with Ena/VASP family members to regulate actin assem