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First published online 15 March 2005
doi: 10.1242/jcs.02285

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WASP-related proteins, Abi1 and Ena/VASP are required for Listeria invasion induced by the Met receptor

¹ Unité des Interactions Bactéries-Cellules, Institut Pasteur, INSERM U604, INRA USC2020, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France
² Division of Cancer Genomics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, MInato-ku, Tokyo 108-8639, Japan, and PRESTO, Japan Science and Technology Agency (JST)
³ The European Institute of Oncology (IEO) and the FIRC Institute for Molecular Oncology (IFOM), Via Adamello 16, 20139 Milan, Italy
⁴ Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, Cambridge, MA 02139-4307, USA
⁵ Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

View larger version (88K): [in a new window]   Fig. 1. WAVE2 is localized at membrane ruffles and phagocytic cups in Vero cells. (A) Immunoblots of Vero cell lysates using anti-pan WAVE and anti-N-WASP antibodies (upper panel), and anti-WAVE1, anti-WAVE2 and anti-pan-WAVE antibodies (lower panel). N-WASP is detected as a protein of 65 kDa and WAVE at 80 kDa and 90 kDa, the latter reflecting phosphorylated WAVE. Actin was detected as a loading control. (B) Colocalisation of WAVE and actin filaments in areas of ruffling (indicated by arrows) in Vero cells stimulated with 4.5 nM InlB. N-WASP is not localized at InlB-induced membrane ruffles. Cells were stained with anti-pan WAVE or anti-N-WASP antibodies and phalloidin-488. (C) Recruitment of WAVE but not N-WASP to bacteria-induced phagocytic cups in Vero cells. The accumulation of WAVE and actin filaments is indicated by arrows. Cells were stained with anti-pan WAVE or anti-N-WASP antibody (red), anti-InlA to detect bacteria (blue) and phalloidin-488 (green). Bar, 5 µm (B) and 2 µm (C).

View larger version (84K): [in a new window]   Fig. 2. WAVE2-GFP recruitment during InlB-mediated internalisation and ruffling in Vero cells. (A) Recruitment of WAVE2-GFP at InlB-induced ruffles. Vero cells, transiently transfected with WAVE2-GFP, untreated (resting cells) or stimulated with 4.5 nM InlB (+InlB) and stained with phalloidin-546 to detect F-actin. (B) Recruitment of WAVE2-GFP at bacteria-induced phagocytic cups at different stages of the internalisation process. Vero cells were transiently transfected with WAVE2-GFP or N-WASP-GFP. Total bacteria associated with cells were visualized in phase contrast and bacteria that are fully or partly extracellular were identified by labelling with anti-Listeria antibodies prior to cell permeabilisation. F-actin was detected with phalloidin-546. In the merged images (right-hand panels), extracellular bacteria are blue, F-actin is green and the GFP-tagged proteins are red. a, an extracellular bacterium not associated with WAVE-GFP; b, an extracellular bacterium associated with WAVE2-GFP and low amounts of actin filaments; c, an extracellular bacterium; d, an intracellular bacterium in an F-actin-rich phagocytic cup to which WAVE2-GFP localizes. N-WASP-GFP is not recruited to bacteria in F-actin phagocytic cups. Bar, 10 µm (A) and 2 µm (B).

View larger version (32K): [in a new window]   Fig. 3. Inhibition of Rho-GTPases and WASP-related proteins by dominant-negative constructs differentially affects invasion in Vero and HeLa cells. The percentage of intracellular bacteria was quantified in cells, non transfected (NT) or transiently transfected with the indicated constructs. The percentage in non-transfected cells has been assigned a value of 100. The level of entry in transfected cells is given as a relative value. Values are the mean±s.d. of at least three independent experiments.

View larger version (60K): [in a new window]   Fig. 4. Expression of WASP-related proteins in HeLa cells and recruitment of N-WASP at phagocytic cups. (A) HeLa, WAVE2-knockdown (KD) and Abi1-knockdown cell lysates were subjected to western blotting with anti-WAVE1, anti-WAVE2 and anti-actin. WAVE1 is overexpressed in WAVE2-knockdown cells. WAVE1 and WAVE2 protein amounts are strongly reduced in Abi1-knockdown cells. (B) N-WASP is localized at phagocytic cups in HeLa cells. Cells were stained with anti-N-WASP antibodies, DAPI to detect bacteria, and phalloidin-488 (red, blue and green, respectively, in the merged images in the right-hand panels). a, a bacterium not associated with N-WASP; b, a bacterium associated with N-WASP and F-actin-rich phagocytic cups; c, a bacterium associated with N-WASP and no actin filaments. (C) NWASP-GFP is recruited at bacterial entry site (arrows). Bacteria are labelled with DAPI. Bar, 2 µm.

View larger version (32K): [in a new window]   Fig. 5. WAVE proteins, N-WASP, Abi-1 and VASP siRNA knockdown block Listeria entry into HeLa cells. (A) Silencing of WASP-related proteins and VASP by siRNA. Suppression of gene expression was analysed in control HeLa or in WAVE2-knockdown or Abi1-knockdown cells, by immunoblotting with antibodies against WAVEs isoforms, N-WASP, VASP or actin. (B) Invasion assays in siRNA-transfected cells. The percentage of intracellular bacteria were quantified by gentamicin assays in cells, non-transfected (NT) or transiently transfected, or co-transfected, with the indicated siRNA. The percentage in non-transfected cells was assigned a value of 100. The level of entry in transfected cells is given as a relative value. Values are the mean±s.d. of at least three independent experiments. CT, control siRNA; NW, N-WASP; Rd, random siRNA; W1, WAVE1; W2, WAVE2.

View larger version (82K): [in a new window]   Fig. 6. Inactivation of WAVE1 or co-inactivation of WAVE1 together with N-WASP in WAVE2-knockdown cells profoundly affects HGF-induced actin cytoskeleton rearrangements. (A) HGF (0.6 nM) stimulation induces formation of peripheral (P) and dorsal (D) ruffles in HeLa cells. (B) WAVE2 knockdown (K.D.) HeLa cells were non-transfected (NT) or transfected with WAVE1 siRNA (W1) or co-transfected with WAVE1 and N-WASP siRNA (W1+NW), and stimulated or not with HGF, fixed and stained with phalloidin-488 to detect F-actin. Resting cells display no apparent morphological alterations. Ruffles are mainly formed in WAVE2-knockdown cells. Filopodia are mainly formed in WAVE2-knockdown cells treated with WAVE1 siRNA. Cells detach from each other in WAVE2-knockdown cells treated with WASP1 + NWASP siRNA. Squared regions indicate the position of the fields magnified below. D, dorsal; J, cell junctions; P, peripheral. Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 7. A role for Abi1 in InlB-induced processes. (A) Colocalisation of Abi1 with F-actin at phagocytic cups. Vero cells were stained with Abi1 and anti-Listeria antibodies and phalloidin-488. Arrows show recruitment of endogenous Abi1 at an F-actin-rich phagocytic cup. (B) Abi1 gene silencing impairs the formation of InlB-induced membrane ruffles. Control HeLa or Abi-knockdown cells were untreated (resting cells) or stimulated with 4.5 nM InlB (+InlB) and stained with Abi1 antibody and Phalloidin-488 to detect F-actin. Abi1 colocalizes with F-actin at membrane ruffles (arrows). Ruffles are inhibited in Abi-knockdown cells. Bar, 2 µm (A) and 10 µm (B).

View larger version (59K): [in a new window]   Fig. 8. VASP and Mena-GFP recruitment at bacteria-induced phagocytic cups. (A) Cells were stained with anti-InlA antibody to detect bacteria (blue), Phalloidin-488 to detect F-actin (green) and anti-VASP antibody (red). (B) Overexpression of Mena increases the F-actin content of phagocytic cups. Vero cells transiently transfected with Mena-GFP were stained with anti-Listeria antibody (blue) and phalloidin-546 to detect F-actin (green). Mena-GFP is shown in red. Phagocytic cups at the site of entry present a thicker actin network (arrows), magnified in the boxed region. Merged images are shown in bottom right-hand panels. Bar, 1 µm (A) and 2 µm (B).

View larger version (29K): [in a new window]   Fig. 9. Inhibition of invasion and ruffling upon Ena/VASP sequestration. The percentage of intracellular InlB-coated latex beads or bacteria, as well as the percentage of cells forming ruffles upon InlB stimulation, was determined in Vero cells, non transfected (NT) or transiently transfected with APPPP-Mito or FPPPP-mito. The percentage in non-transfected cells has been assigned a value of 100. The level of entry or of ruffling in transfected cells is given as a relative value. Values are the mean±s.d. of at least three independent experiments.

View larger version (71K): [in a new window]   Fig. 10. Modulation of Ena/VASP activity affects the structure of InlB-mediated phagocytic cups. Vero cells, non transfected (NT) or transiently transfected with Mena-GFP, FPPPP-mito-GFP or APPPP-mito-GFP, were incubated with InlB-coated beads. In the merged images, F-actin is green, GFP is red, and InlB-beads or bacteria are blue. In cells overexpressing Mena (column 2), phagocytic cups around InlB-coated beads (in blue) present a thicker actin network (large arrow) compared to controls (actin cups in non-transfected cells; column 1). Conversely, in cells in which Ena/VASP is sequestered at the mitochondria (FPPPP-Mito, column 3), phagocytic cups seem to contain a less dense actin meshwork (thin arrows) when compared to controls (non-transfected cells, NT, column 1 and APPPP-Mito, column 4). Bar, 2 µm.

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