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.

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