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First published online 26 April 2005
doi: 10.1242/jcs.02351

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ARF6 GTPase controls bacterial invasion by actin remodelling

María Eugenia Balañá¹, Florence Niedergang^2,*, Agathe Subtil^1,*, Andrés Alcover¹, Philippe Chavrier² and Alice Dautry-Varsat^1,

¹ Unité de Biologie des Interactions Cellulaires, Institut Pasteur, CNRS URA 2582, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
² CNRS UMR 144 Institut Curie, 12 rue Lhomond, 75005 Paris, France

View larger version (30K): [in a new window]   Fig. 1. ARF6 recruitment and distribution during bacterial entry. Cells transfected with HA-tagged-ARF6 WT were infected for 5 minutes with FITC-coupled C. caviae GPIC (white spots). Cells were fixed and stained with anti-HA antibody and Cy5-labelled secondary antibody (top panel) and with Alexa 546-coupled phalloidin (middle panel). The bottom panel is the merged image of ARF6 (red), actin (green) and bacteria (white) staining. A medial confocal section is shown. x-z and y-z optical sections on the positions marked by the crossed lines are displayed on the top and right of the image. ARF6 recruitment is observed at a site of intense actin polymerization, where a bacterium is present (arrows). Bar, 5 µm.

View larger version (57K): [in a new window]   Fig. 2. Inhibition of ARF6 impairs infection. (A-C) ARF6 T27N inhibits C. caviae GPIC infection. HeLa cells transiently expressing the HA-tagged-ARF6 WT or ARF6 T27N were infected for 18 hours. Cells were then fixed and stained with anti-HA antibody followed by TRITC-coupled anti-rat antibody to detect transfected cells (arrows). Bacteria were revealed using FITC-conjugated anti-Chlamydia antibody (A). Arrowheads indicate bacterial inclusions. The efficiency of infection (B) or bacterial association with cells (C) was calculated as indicated in the Methods section and the efficiency of infection or association in transfected cells relative to that in surrounding non-transfected cells (NT) is plotted. The results are the mean±s.e.m. of four independent experiments. (D) ARF6 T27N inhibits bacterial entry. HeLa cells were transfected with the indicated plasmids and infected on the following day for 4 hours. Extracellular bacteria were labelled with anti-Chlamydia antibody followed by a Cy5-coupled secondary antibody. The cells were then permeabilized in 0.05% saponin and incubated with FITC-conjugated anti-Chlamydia antibody to label all bacteria. Transfected cells were visualized with anti-HA antibody followed by TRITC-coupled secondary antibody. The number of surface-associated and intracellular bacteria was counted in the transfected and non-transfected population (n>50 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells. Data are the mean±s.e.m. of four independent experiments. (E-F) Bacterial entry is impaired in ARF6-depleted cells. HeLa cells were treated with ARF6 siRNA prior to measuring bacteria entry as in D. Intracellular bacteria appear green; surface-associated bacteria appear red or yellow. Quantification is shown in F. The efficiency of entry is expressed relative to that in non-treated cells. One representative experiment out of four is shown. (G) Effect of RNAi on ARF6 protein expression. Equal amounts of protein from control or siRNA-treated cell lysates were run on SDS-PAGE and immunoblots were probed with anti-ARF6 antibodies. The western blot corresponds to the experiment shown in F.

View larger version (49K): [in a new window]   Fig. 3. ARF6 activation upon infection. HeLa cells in suspension were centrifuged for 20 seconds with or without bacteria (control) and incubated for the indicated times at 37°C as described in the Methods section. The cells were then lysed on ice and incubated with the ARF binding domain of ARHGAP 10 (ARF-BD) fused to GST, which binds the active GTP-bound form of ARF6. The proteins associated with this GST construct were pulled down using glutathione sepharose and analysed by western blot using anti-ARF6 antibodies. Aliquots of total cell lysates were immunoblotted for total ARF6, showing that the total amount of protein was identical at all time points. Data are representative of five experiments.

View larger version (69K): [in a new window]   Fig. 4. ARF6 involvement in actin polymerization. (A) ARF6 QS-EI inhibits bacterial entry. HeLa cells were transfected with the ARF6 QS-EI construct and infected for 4 hours with C. caviae GPIC on the following day. Extracellular and intracellular bacteria, as well as transfected cells, were labelled as described in Fig. 2D. The number of surface-associated and intracellular bacteria was counted in the transfected and non-transfected population (n>25 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells. Data are the mean±s.e.m. of three independent experiments. (B,C) ARF6 T27N and QS-EI mutants affect actin polymerization upon bacteria entry. Cells transfected or not with the ARF6 T27N-HA or the ARF6 QS-EI constructs were infected for 5 minutes as described in the Methods section. Actin was visualized with Texas Red-coupled phalloidin, ARF6 T27N-transfected cells were identified with anti-HA antibody and Alexa 488-labelled second antibody and cells transfected with the ARF6 QS-EI were visualized with anti-ARF6 antibody and Alexa 488-coupled second antibody. A representative experiment is shown in B. The arrows indicate actin patches. The actin patches visualized 5 minutes post-infection at the entry sites were quantified in transfected and non-transfected cells (C). Results are expressed as percent of patches in transfected cells compared with percent of patches in non-transfected cells. Data are the mean±s.e.m. of three independent experiments.

View larger version (59K): [in a new window]   Fig. 5. Recruitment of PIP 5-kinase at the sites of bacterial entry and local PIP2 production. (A) PIP 5-kinase localizes at the sites of bacteria entry. HeLa cells were transfected with HA-tagged PIP 5-kinase for 18 hours and infected with FITC-coupled Chlamydia for 5 minutes. Transfected cells were identified with anti-HA antibody followed by anti-rat TRITC antibody (in red). PIP 5-kinase is shown recruited (arrows) around the bacteria (green) (top left panel). In the bottom panels a higher magnification of the entry sites is shown; note the absence of PIP 5-kinase in the central area (bottom right panel) where the bacterium is present (bottom left panel). Medial confocal optical sections are shown. The overall structure at the sites of bacterial entry can be visualized in the differential interference contrast image (arrows in the top right panel). Results are representative of at least three independent experiments. (B) Distribution of PIP2 during bacterial entry. HeLa cells transfected with GFP-PLC-PH and HA-tagged PIP 5-kinase were infected for 5 minutes as indicated. Cells were fixed and stained with anti-HA antibody followed by anti-rat TRITC to reveal PIP 5-kinase (red). The fluorescence of GFP-PLC-PH reveals the presence of PIP2 (green) (top panel). HeLa cells transfected with GFP-PLC-PH were infected as indicated with Cy5-coupled Chlamydia for 5 minutes before fixation. The arrowheads indicate PIP2 found at the entry sites accumulated around a bacterium (red). The differential interference contrast image with the overall structure is shown (bottom panel). Results are representative of at least three independent experiments. (C) PIP 5-kinase depletion by RNAi impairs bacterial infection. HeLa cells were treated for 48 hours with PIP 5-kinase siRNA and infected for 20 hours prior to quantification of the number of infected cells. Infected cells were visualized with FITC-coupled anti-Chlamydia antibody. The number of infected cells is expressed relative to that in non-treated cells. The results presented are the mean±s.e.m. of three experiments. (D) Transient expression of Lyn-phosphatase affects Chlamydia entry. HeLa cells were transfected to express Lyn-CFP-Inp54p, infected with FITC-coupled bacteria for 4 hours on the following day and bacteria entry was quantified. Extracellular bacteria were stained with anti-Chlamydia antibody followed by Cy5-coupled secondary antibody. Surface-associated (Cy5- and FITC-positive) and intracellular bacteria (FITC-positive) were counted in the transfected and non-transfected population (n>25 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells (NT). Data are the mean±s.e.m. of three independent experiments. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 6. Confocal analysis of Chlamydia entry sites. Sites of Chlamydia entry appear as spatially well-organized membrane structures that accumulate ARF6 and its downstream effectors. (A) HeLa cells transfected with HA-tagged-ARF6 WT were infected for 5 minutes with FITC-coupled Chlamydia (blue). Cells were then stained with anti-HA antibody followed by TRITC-coupled second antibody. Inset shows bacterium (blue) surrounded by ARF6 staining. In the right-hand panel, a plan of the 3D reconstructed structure of ARF6 (red) and internalizing bacterium (blue) is shown. Results are representative of at least three independent experiments. (B) HeLa cells transfected with GFP-PLC PH and HA-tagged-ARF6 WT were infected by Chlamydia for 5 minutes. Transfected cells were detected with a specific anti-HA antibody followed by Cy5-coupled secondary antibody (red) and the fluorescence of GFP (green). Higher magnifications of the entry site (arrowheads) are shown in the right-hand panels. Results are representative of at least three independent experiments. (C) HeLa cells transfected with HA-tagged PIP 5-kinase and ARF6 were infected by Chlamydia for 5 minutes. Cells were stained with anti-ARF6 antibody followed by Alexa488-coupled secondary antibody (green), anti-HA antibody followed by Cy5-coupled secondary antibody (red) and with Alexa 633-coupled phalloidin (blue). A higher magnification of the entry site (arrowhead) is shown in the right-hand panel. (D) Scheme showing the relative localization of ARF6, PIP2 and PIP 5-kinase around the internalizing bacteria. PIP2 staining (green) was the closest to the bacterium and covered most of the surface of the round structure; ARF6 (blue) and PIP 5-kinase (red) staining were preferentially enriched on the edges of the round protrusion, leaving a large unstained area in the centre filled by the bacterium. PIP 5-kinase appears to occupy the largest area that overlapped with PIP2 and ARF6 staining. Bar, 2 µm (A and right-hand panels in C); 10 µm (B); 5 µm (left-hand panels in C).