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
Author for correspondence (e-mail: gerke{at}uni-muenster.de)
Accepted September 26, 2001
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SUMMARY |
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Key words: Actin organization, Calcium, Membrane microdomain, Raft
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Introduction |
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Annexins comprise a multigene family of bimodular Ca2+-binding proteins that interact with acidic phospholipids preferentially found at the cytosolic face of cellular membranes (Gerke and Moss, 1997). This interaction is mediated by the annexin core domain, a Ca2+- and lipid-binding module consisting of four or eight homology segments, the annexin repeats. The second principal module in each annexin protein is the N-terminal domain, which is highly variable in sequence and length and specifies individual annexin properties. N-terminal domains of annexins can harbour binding sites for cellular protein ligands and also play a role in specifying the intracellular target membrane of a given annexin, i.e. the membrane to which an annexin is located at resting or elevated Ca2+ levels. Target membranes for annexins include the plasma membrane as well as membranes of the endosomal and the secretory apparatus (Creutz, 1992; Gerke and Moss, 1997; Gruenberg and Emans, 1993; Raynal and Pollard, 1994).
In addition to binding to cellular membranes, several annexins also interact in a Ca2+-regulated manner with elements of the actin cytoskeleton, in particular F-actin and spectrin. The actin-binding sites mapped within annexins so far are also found in the protein core (Filipenko and Waisman, 2001; Jones et al., 1992) and it is not yet known whether phospholipid and F-actin binding are mutually exclusive. However, at least in the case of annexins 2 and 6, membrane and actin-cytoskeleton binding could occur simultaneously, thus allowing for a bridging function. Annexin 6 is the only member of the family comprising eight annexin repeats, which are folded into two separate core modules, each having the potential of binding to a membrane surface or the cytoskeleton (Avila-Sakar et al., 2000; Benz et al., 1996). Annexin 2, however, is a four-repeat annexin but it can form a heterotetrameric complex with its specific ligand, the S100A10 protein. In this complex, a central S100A10 dimer links two annexin 2 chains in a highly symmetrical manner, thereby providing a physical connection between two membrane- and F-actin-binding cores (Gerke and Weber, 1985; Lewit-Bentley et al., 2000; Rety et al., 1999).
Both annexin 2 and 6 are found associated with membranes of the endosomal system and the plasma membrane (Gerke and Moss, 1997). This association appears to occur preferentially at sites of membrane microdomains rich in cholesterol, glycosphingolipids and glycosyl phosphatidylinositol (GPI)-anchored proteins (Babiychuk and Draeger, 2000; Harder and Gerke, 1994; Harder et al., 1997; Oliferenko et al., 1999). Such microdomains, also known as rafts, represent lateral lipid assemblies serving as signalling platforms in a number of cellular processes including T-cell receptor and Ras signalling (Simons and Toomre, 2000). Although the characteristic lipid components of rafts are elements of the exoplasmic leaflet, less is known about lipids enriched in the inner leaflet of such microdomains and their interaction with the cortical cytoskeleton. Annexin 2 appears to be one of the few structural proteins located specifically at this raft-cytoskeleton interface, at least in polarized mammary epithelial and smooth muscle cells (Babiychuk and Draeger, 2000; Oliferenko et al., 1999).
Signalling through the plasma membrane to the underlying actin cytoskeleton also occurs when certain microorganisms attach to the surface of their host cells. Such events are particularly evident in the case of enteropathogenic Escherichia coli (EPEC) diarrhoea-causing pathogens that form microcolonies on intestinal epithelial cells of the infected host. Once attached, the bacteria use a specialized secretion system to deliver into the host cell a number of E. coli-secreted proteins (Esps), which include a receptor for the EPEC outer membrane protein intimin. The translocated intimin receptor (Tir) not only mediates intimate bacterial adhesion but also recruits several components that regulate the actin cytoskeleton including -actinin, ezrin, Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein 2 and 3 (Arp 2/3) complex. This recruitment ultimally leads to the localized loss of microvilli and the formation of actin-rich pedestals beneath the attached bacteria (Frischknecht and Way, 2001; Goosney et al., 2000).
To analyze whether cholesterol-rich membrane domains and cytoplasmically associated annexins could participate in the attachment of EPECs, we localized different annexins as well as green fluoresecent protein (GFP)-tagged GPI and plasma membrane cholesterol in EPEC-infected HeLa cells. We show that annexin 2 concentrates at sites of attached EPEC clusters and that the membrane beneath such clusters is rich in cholesterol and GPI-anchored GFP. This indicates that raft components and annexin 2 attached to the inner leaflet of rafts are involved in initiating rearrangements of the actin cytoskeleton that occur upon EPEC contact.
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Materials and Methods |
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Cell culture and transfections
HeLa cells were maintained in Dulbeccos modified Eagles medium (DMEM) with 10% fetal calf serum, glutamine and antibiotics in a 7% CO2 incubator at 37°C. For transient transfections, cells were grown on coverslips in 35 mm dishes and then transfected with Effectene (Qiagen, Hilden, Germany) using 0.4 µg DNA per well. Cells were used for infection with EPEC 24 hours after transfection.
Bacterial culture and infection
EPEC strains used in this study were EPEC 2348/69 (wild-type), EPEC 2348/69 SE896 (EPECtir, a mutant expressing a C-terminally truncated Tir protein not inserted into the host cell membrane or cytoplasm) (Elliott et al., 1999) and EPEC 2348/69 CVD452 (a mutant defective in type III-dependent secretion) (Jarvis et al., 1995). All EPEC strains were grown in Standard I-medium (Difco, Augsburg, Germany) at 37°C in overnight cultures with shaking at 150 rpm. HeLa cells grown on cover slips were infected with an overnight EPEC culture (approximately 100 bacteria per HeLa cell) in DMEM, 2% fetal calf serum, 1 mM glutamine, 10 mM HEPES and 1% methyl--D-mannose for 3 hours at 37°C in a 10% CO2 incubator. Subsequently, cells were washed intensively with PBS.
Fluorescence microscopy
To visualize endogenous annexin 2, EPEC-infected HeLa cells were permeabilized for 5 minutes on ice with 0.2% Triton-X-100 in PBS containing Ca2+ and Mg2+ (Dulbeccos PBS/Ca2+/Mg2+; BioConcept, Umkirch, Germany) and then fixed in 4% paraformaldehyde (PFA) in the same buffer for 10 minutes at room temperature (RT). Following fixation and washing, cells were treated with 50 mM NH4Cl in PBS to quench free aldehydes and then incubated with undiluted hybridoma supernatant containing the monoclonal anti-annexin 2 antibody HH7 (Thiel et al., 1992) for 45 minutes at RT. Following washing with PBS, secondary Cy2-labelled goat anti-mouse antibodies (Dianova, Heidelberg, Germany), diluted in PBS containing 2% BSA, were applied in the same way.
To analyze cells transfected with the different GFP or YFP fusion constructs, fixation was carried out with 4% PFA in PBS for 10 minutes. For cholesterol labelling, cells were incubated in PBS containing 2% BSA and 50 µg/ml filipin III (Sigma). Filamentous actin was stained with rhodamine-conjugated phalloidin, which was added either during incubation with the secondary antibody or with filipin. Coverslips were mounted in mowiol with 4% n-propyl-gallate as antifade agent and the cells were inspected using a DM RXA fluorescence microscope (Leica, Wetzlar, Germany). Confocal images were obtained using a TCS NT confocal laser scanning microscope (Leica, Wetzlar, Germany).
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Results |
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HeLa cells infected with EPEC for 3 hours showed the characteristic reorganization of the actin cytoskeleton beneath microcolonies of adhering bacteria. Visualization of endogenous annexin 2 with the monoclonal antibody HH7 revealed that the protein accumulates at the sites of actin polymerization (Fig. 1A-C). By contrast, annexins 1 and 3, which had not been located to membrane/cytoskeleton contacts before, failed to be enriched at sites where EPEC attach, thus indicating that the recruitment of annexin 2 is specific (not shown). When inspected more closely, it appears that the annexin 2 staining is not uniform throughout the actin-rich pedestals but that it surrounds pedestals formed underneath individual bacteria in the juxtamembrane region (insets in Fig. 1).
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To analyze the annexin 2 distribution in living cells, we had previously employed an annexin 2-GFP fusion protein. When expressed in HeLa cells, this annexin 2-GFP chimera is found uniformly distributed along the entire plasma membrane with a diffuse fluorescence signal also being present in the cytoplasm (Rescher et al., 2000). The same GFP chimera was employed in the current study to circumvent the potential problem of causing an artificial mislocalization of annexin 2 when differing Ca2+ concentrations were present in the permeabilization medium. Fig. 2 (left panels, A,C,E,G) shows that EPEC attachment induces a recruitment of annexin 2-GFP to the attachment sites that is indistinguishable from that observed for the endogenous protein in cells permeabilized with Triton X-100 in the absence of EDTA. This indicates that the recruitment observed occurs at Ca2+ levels met in the infected cells and is not a phenomenon induced only following Triton X-100 permeabilization in the presence of Ca2+.
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Tir is not required for the targeting of annexin 2 to EPEC attachment sites
Following EPEC adherence, several virulence factors that are encoded on a chromosomal pathogenicity island, the locus of enterocyte effacement (LEE), are transferred into the infected host cell by means of a type III secretion system (Jarvis et al., 1995). One of the proteins transferred, Tir, is inserted into the host plasma membrane and acts as a recruitment factor for a number of actin-regulating proteins including WASP and Arp 2/3 (Frischknecht and Way, 2001; Goosney et al., 2000). This recruitment triggers pedestal formation, which has been shown to depend on phosphorylation of Tir at tyrosine 474 (Kenny, 1999). In line with this crucial role of Tir in inducing pedestal formation, a mutant EPEC strain (EPECtir, SE896), which only expresses a C-terminally truncated Tir protein comprising the N-terminal 233 residues (Elliott et al., 1999), is not capable of inducing pedestals upon attachment (S.L. and M.A.S., unpublished).
To determine whether annexin 2 translocation to the sites of EPEC attachment is mediated via Tir, we made use of this mutant strain. HeLa cells expressing annexin 2-GFP were infected with EPECtir (SE896) for 3 hours and then analyzed for GFP and rhodamine-phalloidin fluorescence. Fig. 3 (left panels, A,C,E,G) shows that, although pedestal formation is compromised in tir mutant-infected HeLa, some F-actin accumulation continues to occur at sites of bacterial contact. However, although the phalloidin signal is significantly diminished in the case of the tir mutant as compared to wild-type EPEC, annexin 2-GFP continues to concentrate at EPECtir attachment sites. It even appears that the extent of the annexin 2-GFP recruitment is more pronounced in the case of tir mutant as compared to wild-type bacteria. Next, we carried out infections with an EPEC mutant strain (CVD452) deficient in secretion of all type III-secreted proteins like, for example, the secreted Esps and Tir (Jarvis et al., 1995). Although adhering CVD mutant bacteria continue to be visible on the infected HeLa cells, they fail to induce the recruitment of annexin 2-GFP (Fig. 3, right panels, B,D,F,H). Thus, the translocation of annexin 2 to sites of EPEC attachment occurs independently of Tir being inserted into the host cell membrane but requires (a) factor(s) encoded on the LEE pathogenicity island.
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Discussion |
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Annexin 2 is a Ca2+-regulated phospholipid and F-actin-binding protein (Gerke and Moss, 1997), and the somewhat reduced recruitment of the Ca2+-defective CM annexin 2 mutant to sites of EPEC attachment suggests that this process requires Ca2+ binding to annexin 2. Although the role of Ca2+ signalling in EPEC-induced actin rearrangement is not clearly established, our data employing a GFP-tagged version of annexin 2 indicate that the Ca2+ concentrations met in EPEC-infected HeLa are sufficient to trigger annexin 2 accumulation beneath EPEC microcolonies. Thus, the accumulation could reflect a Ca2+-dependent binding of the protein to membrane components or F-actin enriched at EPEC attachment sites, or both. The annexin 2 ligand S100A10 is also found enriched at sites of bacterial adherence indicative of the presence of the heterotetrameric annexin 2-S100A10 complex. This complex represents an entity capable of binding phospholipids and F-actin simultaneously and thus could serve as a bridging function between the host cell membrane and the rearranged actin cytoskeleton beneath EPEC microcolonies. Such linking function is supported by the observation that annexin 2 appears to be concentrated around the actin-rich pedestals in close proximity to the plasma membrane (Fig. 1).
Which membrane component(s) could specify the recruitment of annexin 2 to the sites of EPEC attachment? Tir, a bacterial protein incorporated into the host cell membrane, can be excluded because a Tir-defective mutant strain is still capable of inducing a strong annexin 2 accumulation. However, other bacterial Esps, in particular those discussed to be inserted into the host membrane (DeVinney et al., 1999; Wachter et al., 1999), could represent anchors for annexin 2, given that they are produced in the EPECtir strain (Elliott et al., 1999). Likewise, it is possible that membrane components of the host are involved. In uninfected HeLa cells annexin 2 assumes, at least at the light microscopical level, a uniform distribution along the plasma membrane and in the cortical cytoskeleton (Rescher et al., 2000; Thiel et al., 1992). Thus, it seems plausible that annexin 2-binding structure(s) are distributed over the entire membrane in noninfected cells and only become concentrated when EPEC or EPECtir adhere. Our analyses identify cholesterol and GPI-anchored proteins as membrane components of the host cell that accumulate at EPEC attachment sites. Although a direct binding of annexin 2 to cholesterol or GPI anchors has not been reported, it is evident from a number of studies that the protein can associate with rafts or raft-like structures rich in cholesterol and GPI-anchored proteins. In BHK cells, membrane-bound annexin 2 can be released specifically upon cholesterol sequestration, and in mammary epithelial cells, as well as BHK and smooth muscle cells, the protein is significantly enriched in the raft fraction in the presence of Ca2+ (Babiychuk and Draeger, 2000; Harder and Gerke, 1994; Oliferenko et al., 1999). Although the raft component acting as the actual binding partner still awaits identification, our findings suggest that EPEC attachment is accompanied by a clustering of rafts, which in turn is responsible for and could be stabilized by an enrichment of annexin 2 at such sites.
By employing an EPEC strain defective in Tir we could show that both accumulation of raft components and annexin 2 does not require the incoporation of Tir into the host cell membrane. However, Tir has been shown to be essential for pedestal formation, which depends on N-WASP recruitment and Arp 2/3 activation (Frischknecht and Way, 2001; Goosney et al., 2000). It is therefore tempting to speculate that, during initial Tir-independent adherence, EPEC interact with one or more raft components of the host membrane, thereby inducing raft clustering. Although not shown for EPEC so far, binding of bacterial pathogens to raft components, in particular GPI-anchored proteins, is not without precedent. Among other things, it has been shown that diffusely adhering E. coli bind via the fimbrial adhesin F1845 to the GPI-anchored decay accelerating factor (Pfeiffer et al., 1998) and that the pore-forming aerolysin produced by Aeromonas hydrophila binds to a GPI-anchored protein receptor (Pfeiffer et al., 1998). EPEC-induced clustering of rafts could represent a signal for actin rearrangement, which is required prior to the insertion of Tir and the actual pedestal formation. In line with this hypothesis, the Tir-defective strain triggers some actin rearrangement at sites of bacterial attachment without being capable of inducing the characteristic pedestals. Similar to what was proposed for Ca2+-regulated membrane segregation in smooth muscle cells (Babiychuk and Draeger, 2000), annexin 2 could participate in this scenario by stabilizing the formation of larger raft patches beneath adhering EPEC. By this means annexin 2 may act at an early stage of bacterial infection and possibly independently of actin pedestal formation. The view that EPEC can utilize two separate ways of inducing actin polymerization, one depending on Tir-dependent N-WASP recruitment and the other on raft clustering accompanied by annexin 2 accumulation, is supported by our finding that infection with wild-type EPEC of a liver cell line (HepG2), which expresses only trace amounts of annexin 2, still induces actin rearrangement at sites of bacterial attachment (data not shown). The action of annexin 2 inferred from the cell culture studies described here could be of physiological relevance during EPEC infections occurring in the intestine. Both annexin 2 and S100A10 are particularly abundant in intestinal epithelial cells where they show an enrichment in the apical membrane cytoskeleton below the level of the microvilli (Gerke and Weber, 1984). This localization could facilitate a rapid action of annexin 2 in reorganizing the apical membrane/cytoskeleton following EPEC contact. Future experiments have to define the precise site of annexin 2 action and have to identify the types of EPEC infections that require annexin 2.
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ACKNOWLEDGMENTS |
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