Suzuki T , Mimuro H , Miki H , Takenawa T , Sasaki T , Nakanishi H , Takai Y , Sasakawa C .

	Figure 1. Effects of the dominant-active Rho family GTPases on the speed of Shigella motility in Swiss 3T3 cells. Relative velocity values before and after microinjection with (A) Cdc42-G12V; (B) Rac1-G12V; and (C) RhoA-G14V. Arrows indicate the time of microinjection. •, bacteria moving at low speeds (<5 μm/min) before microinjection; ○, bacteria moving at high speeds (>5 μm/min) before microinjection. The numbers of intracellular bacteria analyzed for microinjection were: Cdc42-G12V, 32; Rac1-G12V, 22; and RhoA-G12V, 28; in over five experiments. Error bars represent SEM.
	Figure 2. The effect of RhoGDI on the actin assembly from E. coli (VirG) and Listeria in Xenopus egg extracts. (A) RhoGDI was added to the egg extracts and assayed for the effect on the bacterial motility and assembly of actin (see text for details). Black bars, bacterial speeds; gray bars, F-actin intensities. All activities are normalized to untreated extracts. (B) The percentage of bacteria associating actin clouds (black bars) or actin tails (gray bars). Error bars in A and B represent SEM from three separate experiments. (C) Inhibition of the actin assembly by RhoGDI in the extracts as viewed with DAPI-bacteria (blue) and TMR-actin (red): a and b, actin assembly from E. coli (VirG); c and d, actin assembly from Listeria; a and c, untreated extracts; b and d, RhoGDI (at 400 nM). All images were observed 10 min after mixing bacteria with extracts. Bars, 10 μm.
	Figure 3. The effect of Rho family GTPases on the VirG-directed actin assembly in Xenopus egg extracts. (A) GTPγS-charged Cdc42-G12V or Rac1-G12V was added to egg extracts along with RhoGDI (at 400 nM). Cdc42-T17N, a dominant negative mutant of Cdc42, was added in the absence of RhoGDI. Black bars, bacterial speeds; gray bars, F-actin intensities. All activities are normalized to untreated extracts. (B) The percentage of bacteria associating actin clouds (black bars) or actin tails (gray bars). Error bars in A and B represent SEM from three separate experiments. (C) Actin assembly as viewed with DAPI-bacteria (blue) and TMR-actin (red) in the presence of: a, RhoGDI and Cdc42-G12V (GTPγS); b, RhoGDI and Rac1-G12V (GTPγS); and c, Cdc42-T17N. The images were observed 10 min after mixing bacteria with extracts. Bar, 10 μm.
	Figure 4. The effect of RhoGDI on the interaction between N-WASP and VirG in Xenopus egg extracts. (A) Cy2-labeled recombinant N-WASP (at 75 nM) was added to the egg extracts without (a, c, e, and g) or with (b, d, f, and h) RhoGDI (at 400 nM). a and b, DAPI-labeled bacteria; c and d, Cy2-labeled N-WASP; e and f, TMR-actin. The yellow color in the combined image shown in g and h indicates the colocalization between N-WASP (green) and actin (red). The images were observed 10 min after mixing bacteria with extracts. (B) Quantitation of N-WASP intensity, bacterial motility, and F-actin intensity without or with RhoGDI (at 400 nM). White bars, N-WASP intensities; black bars, bacterial speeds; gray bars, F-actin intensities. All activities are normalized to untreated extracts. Error bars represent SEM from three separate experiments. Bar, 10 μm.
	Figure 5. The capacity of N-WASP to interact with Cdc42 is critical for promoting actin assembly in Xenopus egg extracts. (A) Recombinant wild-type (WT) N-WASP (or H208D mutant) (at 45 nM) was added back to the N-WASP–depleted extracts in the absence or presence of RhoGDI (at 400 nM) or Cdc42-G12V (GTPγS) (at 400 nM). Black bars, bacterial speeds; gray bars, F-actin intensities. All activities are normalized to depleted extracts plus wild-type N-WASP. (B) The percentage of bacteria associating actin clouds (black bars) or actin tails (gray bars). Error bars in A and B represent SEM from three separate experiments. (C) Actin assembly as viewed with DAPI-bacteria (blue) and TMR-actin (red) in the N-WASP–depleted extracts in the presence of: a, no addition; b, wild-type (WT) N-WASP; c, wild-type N-WASP and RhoGDI; d, wild-type N-WASP, RhoGDI, and Cdc42-G12V (GTPγS); e, H208D mutant, RhoGDI, and Cdc42-G12V (GTPγS). The images were observed 10 min after mixing bacteria with extracts. Bar, 10 μm.
	Figure 6. Cdc42 stimulates the VirG-induced actin polymerization by N-WASP–Arp2/3 complex in vitro. (A) Inhibition of VirG-induced actin polymerization by RhoGDI. High speed supernatants containing pyrene actin (∼10% labeled) were incubated with buffer (×), 100 nM VirG-α1 (○), 250 nM VirG-α1 (•), 400 nM RhoGDI (▴), or 250 nM VirG-α1 and 400 nM RhoGDI (▵). (B–D) G-actin (2.2 μM, 10% pyrenyl labeled) was induced to polymerize by addition of 0.1 M KCl and 1 mM MgCl2 in the presence or absence of Arp2/3 complex (20 nM) and other components as indicated. (B) VirG-α1 activates N-WASP–Arp2/3 complex. Actin was polymerized alone (×); in the presence of 20 nM Arp2/3 (○); in the presence of Arp2/3 and 0.1 μM VirG-α1 (•); Arp2/3 and 0.25 μM N-WASP (□); Arp2/3, 0.25 μM N-WASP, and 0.1 μM (▪) or 0.5 μM (▵) VirG-α1. (C) Activation of N-WASP–Arp2/3 complex by VirG-α1 is more enhanced in the presence of Cdc42. Actin was polymerized in the presence of Arp2/3 and 0.25 μM N-WASP (×); Arp2/3, N-WASP, and 0.25 μM Cdc42-G12V (GTPγS) expressed by baculovirus (○); Arp2/3, N-WASP, and 0.1 μM VirG-α1 (•); Arp2/3, N-WASP, 0.25 μM Cdc42-G12V (GTPγS), and 0.1 μM (▵) or 0.5 μM (▴) VirG-α1; Arp2/3, 0.25 μM H208D mutant of N-WASP, 0.25 μM Cdc42-G12V (GTPγS), and 0.1 μM VirG-α1 (□). (D) Effect of VirG-α3 on actin polymerization mediated by N-WASP–Arp2/3 complex. Actin was polymerized in the presence of Arp2/3 and 0.25 μM N-WASP (×); Arp2/3, N-WASP, and 0.1 μM VirG-α3 (○); Arp2/3, N-WASP, 0.25 μM Cdc42-G12V (GTPγS), and 0.1 μM (▵) or 0.5 μM (▴) VirG-α3.
	Figure 7. The effect of Cdc42 on the interaction between N-WASP and VirG in vitro. (A) Recombinant histidine-tagged N-WASP (His-N-WASP) was incubated with various concentrations of VirG-α1 or GTPγS-charged Cdc42-G12V. Proteins precipitated with nickel affinity resin were subjected to Western blotting. (B) Recombinant VirG-α1 and N-WASP were incubated with increasing amounts of GTPγS-charged Cdc42-G12V. Immunoprecipitated proteins (IP) with anti-VirG antibody were analyzed by Western blotting.
	Figure 8. In vitro reconstitution of actin assembly on E. coli (VirG) and Listeria surface. (A) E. coli expressing VirG were incubated in F buffer containing 0.4 μM N-WASP, 70 nM Arp2/3 complex, and 1 μM TMR-actin, with or without 0.4 μM Cdc42-G12V (GTPγS) expressed by baculovirus. (B) Listeria were incubated in F buffer containing 70 nM Arp2/3 complex and 1 μM TMR-actin: a, d, and g, DAPI-labeled bacteria; b, e, and h, TMR-actin; c, f, and i, merged images. Insets show magnified images. Bar, 10 μm.
	Figure 9. Localization of Cdc42 in COS-7 cells expressing GFP-Cdc42 infected with Shigella. (A) Phase–contrast image; (B) localization of GFP-Cdc42; (C) localization of F-actin visualized by staining with rhodamine-phalloidin. The yellow color in the combined image (D) indicates colocalization between GFP-Cdc42 (green) and F-actin (red). Arrows and arrowheads show Shigella-associated actin clouds and actin tails, respectively. Bar, 10 μm.
	Figure 10. Actin assembly from Shigella in infected COS-7 cells expressing the H208D mutant. (A) COS-7 cells overexpressing wild-type (WT) N-WASP (a–c) or H208D mutant (d–f) were infected with Shigella and immunostained using FITC-labeled anti–N-WASP antibody (a and d) and rhodamine-phalloidin (b and e). The yellow color in the combined image shown in c and f indicates colocalization between N-WASP (green) and actin (red). The arrowheads indicate intracellular Shigella. (B) The percentage of intracellular bacteria associating actin assembly. Error bars represent SEM from three separate experiments. Bar, 10 μm.
	Figure 11. Cell to cell spreading of Shigella in infected MDCK cells stably expressing wild-type (WT) N-WASP or H208D mutant. (A) Expression of N-WASP variants in stable transfectants of MDCK cell lines. Total cell lysates were subjected to Western blotting with anti–E-cadherin, anti–N-WASP, and antiactin antibodies. Representative data are shown. (B) Dark field photomicrographs of plaque formation in MDCK cells stably expressing N-WASP variants 3 d after infection with Shigella. The results shown are representative of three independent experiments: a, mock-transfected cells; b, wild-type N-WASP; c, H208D mutant. (C) Localization of E-cadherin and ZO-1 in MDCK cells stably expressing N-WASP variants. Mock-transfected cells (a, d, g, and j), wild-type expressing cells (b, e, h, and k), and H208D mutant expressing cells (c, f, i, and l) were double-stained with anti–E-cadherin antibody (a–f) and anti–ZO-1 antibody (g–l). a–c and g–i, junctional levels; d–f and j–l, vertical sections. Bars, (B) 1 mm; (C) 10 μm.

Adam, Rho-dependent membrane folding causes Shigella entry into epithelial cells. 1996, Pubmed

Adam, Rho-dependent membrane folding causes Shigella entry into epithelial cells. 1996, Pubmed
Allaoui, icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. 1992, Pubmed
Bear, SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. 1998, Pubmed
Bernardini, Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. 1989, Pubmed
Carlier, Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection. 1999, Pubmed
Chakraborty, A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells. 1995, Pubmed
Chen, Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear responses. 1996, Pubmed
Cooper, Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. 1983, Pubmed
Cossart, Interactions of Listeria monocytogenes with mammalian cells during entry and actin-based movement: bacterial factors, cellular ligands and signaling. 1998, Pubmed
Cudmore, Actin-based motility of vaccinia virus. 1995, Pubmed
Derry, Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. 1994, Pubmed
Domann, A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. 1992, Pubmed
Dramsi, Intracellular pathogens and the actin cytoskeleton. 1998, Pubmed
Egile, Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility. 1999, Pubmed
Frischknecht, Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. 1999, Pubmed
Frischknecht, Tyrosine phosphorylation is required for actin-based motility of vaccinia but not Listeria or Shigella. 1999, Pubmed , Xenbase
Fu, A salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion. 1999, Pubmed
Fukuda, Cleavage of Shigella surface protein VirG occurs at a specific site, but the secretion is not essential for intracellular spreading. 1995, Pubmed
Goldberg, Unipolar localization and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement. 1993, Pubmed
Gouin, A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii. 1999, Pubmed , Xenbase
Hall, Rho GTPases and the actin cytoskeleton. 1998, Pubmed
Hardt, S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. 1998, Pubmed
Hauck, CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway. 1998, Pubmed
Heinzen, Directional actin polymerization associated with spotted fever group Rickettsia infection of Vero cells. 1993, Pubmed
Kadurugamuwa, Intercellular spread of Shigella flexneri through a monolayer mediated by membranous protrusions and associated with reorganization of the cytoskeletal protein vinculin. 1991, Pubmed
Kocks, L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. 1992, Pubmed
Kouyama, Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. 1981, Pubmed
Laine, Vinculin proteolysis unmasks an ActA homolog for actin-based Shigella motility. 1997, Pubmed
Laurent, Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. 1999, Pubmed
Lechler, In vitro reconstitution of cortical actin assembly sites in budding yeast. 1997, Pubmed
Lett, virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence. 1989, Pubmed
Li, Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton. 1997, Pubmed
Loisel, Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. 1999, Pubmed
Ma, Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts. 1998, Pubmed , Xenbase
Makino, A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. 1986, Pubmed
Marchand, Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface. 1995, Pubmed , Xenbase
Miki, WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. 1998, Pubmed
Miki, Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. 1998, Pubmed
Miki, N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. 1996, Pubmed
Moreau, Cdc42 is required for membrane dependent actin polymerization in vitro. 1998, Pubmed , Xenbase
Mounier, Rho family GTPases control entry of Shigella flexneri into epithelial cells but not intracellular motility. 1999, Pubmed , Xenbase
Niebuhr, 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. 1997, Pubmed
Nobes, Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. 1995, Pubmed
Prévost, Unipolar reorganization of F-actin layer at bacterial division and bundling of actin filaments by plastin correlate with movement of Shigella flexneri within HeLa cells. 1992, Pubmed
Ridley, The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. 1992, Pubmed
Ridley, The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. 1992, Pubmed
Rohatgi, The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. 1999, Pubmed , Xenbase
Rosenblatt, Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails. 1997, Pubmed , Xenbase
Sasakawa, Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a. 1988, Pubmed
Sasakawa, Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. 1986, Pubmed
Self, Purification of recombinant Rho/Rac/G25K from Escherichia coli. 1995, Pubmed
Suetsugu, Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. 1999, Pubmed
Suzuki, Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri. 1994, Pubmed
Suzuki, Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri. 1998, Pubmed , Xenbase
Suzuki, Functional analysis of Shigella VirG domains essential for interaction with vinculin and actin-based motility. 1996, Pubmed , Xenbase
Suzuki, Extracellular transport of VirG protein in Shigella. 1995, Pubmed
Tanaka, Control of reorganization of the actin cytoskeleton by Rho family small GTP-binding proteins in yeast. 1998, Pubmed
Teysseire, Intracellular movements of Rickettsia conorii and R. typhi based on actin polymerization. 1992, Pubmed
Theriot, The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. 1992, Pubmed
Theriot, Listeria monocytogenes-based assays for actin assembly factors. 1998, Pubmed , Xenbase
Tran Van Nhieu, IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. 1999, Pubmed
Van Aelst, Rho GTPases and signaling networks. 1997, Pubmed
Watarai, rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells. 1997, Pubmed
Welch, Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. 1998, Pubmed
Welch, The world according to Arp: regulation of actin nucleation by the Arp2/3 complex. 1999, Pubmed
Welch, Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes. 1997, Pubmed , Xenbase
Zeile, Recognition of two classes of oligoproline sequences in profilin-mediated acceleration of actin-based Shigella motility. 1996, Pubmed