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Cell-surface attachment of pedestal-forming enteropathogenic E. coli induces a clustering of raft components and a recruitment of annexin 2

Nicole Zobiack^1,*, Ursula Rescher^1,*, Sven Laarmann², Silke Michgehl², M. Alexander Schmidt² and Volker Gerke^1,

Institute for Medical Biochemistry,
¹ Center for Molecular Biology of Inflammation and
² Institute of Infectiology University of Münster, von-Esmarch-Str. 56, D-48149 Münster, Germany
^* These authors contributed equally to this work

View larger version (29K): [in a new window]   Fig. 1. Annexin 2 is recruited to sites of adhering EPEC. HeLa cells were infected with EPEC for 3 hours, permeabilized and fixed with PFA. The endogenous annexin 2 was visualized using the monoclonal anti-annexin 2 antibody HH7 and F-actin was stained with rhodamine-conjugated phalloidin. Note that EPEC-infected cells show bright annexin 2 (A) and F-actin (B) signals beneath adhering bacteria (arrows) (a merge of the images is given in C). The enlarged insets show a twofold magnification of the boxed region and reveal that annexin 2 seems to be localized in cuplike structures around the recruited actin. Bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 2. Accumulation of annexin 2-GFP and YFP-tagged S100A10 at sites of EPEC attachment. HeLa cells ectopically expressing annexin 2-GFP (left panels; A,C,E,G) or YFP-S100A10 (right panels; B,D,F,H) were infected with EPEC and then processed for analysis of the fluorescent protein signals (A,B) and rhodamine-phalloidin (C,D), respectively. Merged images of the fluorescent signals are shown in E and F, respectively, whereas G and H give phase contrast images identifying the adherent bacteria. Localization of annexin 2-GFP (A) around polymerized actin (C) at sites of adhering EPEC (G) is indistinguishable from that of endogenous annexin 2. Likewise, YFP-S100A10 (B) is targeted to actin-positive structures (D) beneath EPEC microcolonies (H). The enlarged insets show a 2.5-fold magnification of the boxed region. Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 3. Recruitment of annexin 2 to sites of attached EPEC does not require Tir but depends on other secreted Esps. HeLa cells expressing annexin 2-GFP were infected with mutant EPEC strains either lacking a functional Tir protein (EPECtir SE896; left panels; A,C,E,G) or defective in type III-dependent secretion (CVD452; right panels; B,D,F,H). The annexin 2-GFP staining (A,B) is compared to that of rhodamine-phalloidin (C,D; merged images in E and F). Phase contrast images revealing the adherent bacteria are given in G and H, respectively. The enlarged insets show a 2.5-fold magnification of the boxed region. Note that annexin 2-GFP clearly accumulates beneath adhering EPECtir (SE896), which do not insert Tir into the host cell membrane (A,C,E,G). Mutant EPEC defective in type III secretion, however, fail to induce an annexin 2 recruitment (B,D,F,H). Bar, 10 µm.

View larger version (83K): [in a new window]   Fig. 4. EPEC attachment induces a clustering of raft components. HeLa cells ectopically expressing annexin 2-GFP (A,C,E) or GPI-GFP (B,D,F) were infected with EPECtir (SE896) and then processed for analysis of the fluorescent protein distribution (A,B) or membrane cholesterol distribution by filipin staining (C,D). Phase contrast images revealing the adherent bacteria are given in E and F, respectively. The enlarged insets show a 2.5-fold magnification of the boxed region. Note that both GPI-anchored GFP, as well as annexin 2-GFP, accumulate together with membrane cholesterol at sites of EPEC attachment. Panels G and H show controls of uninfected HeLa cells stained with filipin (G) or transfected with the GPI-GFP expression construct and processed for fluorescent protein analysis (H). Bar, 10 µm.