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Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology

2002; Springer Nature; Volume: 21; Issue: 19 Linguagem: Inglês

10.1093/emboj/cdf522

1460-2075

Kirsten Niebuhr, Sylvie Giuriato, Thierry Pédron, Dana J. Philpott, Frédérique Gaits‐Iacovoni, Julia Sable, Michael P. Sheetz, Claude Parsot, Philippe Sansonetti, Bernard Payrastre,

Mycobacterium research and diagnosis

Article1 October 2002free access Conversion of PtdIns(4,5)P2 into PtdIns(5)P by the S.flexneri effector IpgD reorganizes host cell morphology Kirsten Niebuhr Kirsten Niebuhr Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Sylvie Giuriato Sylvie Giuriato INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France Search for more papers by this author Thierry Pedron Thierry Pedron Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Dana J. Philpott Dana J. Philpott Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Frédérique Gaits Frédérique Gaits INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France Search for more papers by this author Julia Sable Julia Sable Department of Biological Sciences, PO Box 2408, Columbia University, Sherman Fairchild Center, 1212 Amsterdam Avenue, New York, NY, 10027 USA Search for more papers by this author Michael P. Sheetz Michael P. Sheetz Department of Biological Sciences, PO Box 2408, Columbia University, Sherman Fairchild Center, 1212 Amsterdam Avenue, New York, NY, 10027 USA Search for more papers by this author Claude Parsot Claude Parsot Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Philippe J. Sansonetti Philippe J. Sansonetti Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Bernard Payrastre Corresponding Author Bernard Payrastre INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France Search for more papers by this author Kirsten Niebuhr Kirsten Niebuhr Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Sylvie Giuriato Sylvie Giuriato INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France Search for more papers by this author Thierry Pedron Thierry Pedron Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Dana J. Philpott Dana J. Philpott Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Frédérique Gaits Frédérique Gaits INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France Search for more papers by this author Julia Sable Julia Sable Department of Biological Sciences, PO Box 2408, Columbia University, Sherman Fairchild Center, 1212 Amsterdam Avenue, New York, NY, 10027 USA Search for more papers by this author Michael P. Sheetz Michael P. Sheetz Department of Biological Sciences, PO Box 2408, Columbia University, Sherman Fairchild Center, 1212 Amsterdam Avenue, New York, NY, 10027 USA Search for more papers by this author Claude Parsot Claude Parsot Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Philippe J. Sansonetti Philippe J. Sansonetti Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Bernard Payrastre Corresponding Author Bernard Payrastre INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France Search for more papers by this author Author Information Kirsten Niebuhr1, Sylvie Giuriato2, Thierry Pedron1, Dana J. Philpott1, Frédérique Gaits2, Julia Sable3, Michael P. Sheetz3, Claude Parsot1, Philippe J. Sansonetti1 and Bernard Payrastre 2 1Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, cedex 15, France 2INSERM, Unité 563, Centre de Physiopathologie Toulouse Purpan, Departement d'Oncogenèse et signalisation dans les cellules haematopoïetiques, IFR 30, Hôpital Purpan, 31059 Toulouse, cedex, France 3Department of Biological Sciences, PO Box 2408, Columbia University, Sherman Fairchild Center, 1212 Amsterdam Avenue, New York, NY, 10027 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5069-5078https://doi.org/10.1093/emboj/cdf522 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Phosphoinositides play a central role in the control of several cellular events including actin cytoskeleton organization. Here we show that, upon infection of epithelial cells with the Gram-negative pathogen Shigella flexneri, the virulence factor IpgD is translocated directly into eukaryotic cells and acts as a potent inositol 4-phosphatase that specifically dephosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] into phosphatidylinositol 5-monophosphate [PtdIns(5)P] that then accumulates. Transfection experiments indicate that the transformation of PtdIns(4,5)P2 into PtdIns(5)P by IpgD is responsible for dramatic morphological changes of the host cell, leading to a decrease in membrane tether force associated with membrane blebbing and actin filament remodelling. These data provide the molecular basis for a new mechanism employed by a pathogenic bacterium to promote membrane ruffling at the entry site. Introduction Intracellular pathogens have evolved several mechanisms to hijack different cellular systems in the host to promote their entry, including modifications of the actin cytoskeleton and the exploitation of various signalling pathways (Galan and Bliska, 1996). A key factor of their virulence is invasion of enterocytes, which are cells that in normal circumstances are non-phagocytic. Two strategies, the so-called 'zipper' and 'trigger' mechanisms, are used by invasive bacteria to induce their uptake. Some pathogens, such as Listeria, express surface proteins that bind to eukaryotic receptors and lead to membrane zippering around the bacterium (Mengaud et al., 1996). Other pathogens, like Salmonella and Shigella, use a type III secretion system (Van Gijsegem et al., 1993) to inject effector proteins into the host cell and trigger their uptake via membrane ruffles in a process resembling macropinocytosis (Finlay and Falkow, 1990; Francis et al., 1993; Adam et al., 1995). The formation of these entry structures is the result of a complex cross-talk between injected bacterial proteins and components of the target cell that is not yet fully understood. Shigella flexneri is a facultative intracellular pathogen that is the causative agent of bacillary dysentery in humans. Several proteins are secreted by the S.flexneri type III secretion apparatus during growth of bacteria in culture media. According to the current model of the type III secretion pathway, these proteins are potential effectors that might be translocated into epithelial cells upon contact of bacteria with the cell surface. However, because of the rapid uptake of S.flexneri by the cell and its intracytoplasmic lifestyle, no direct translocation of a secreted protein has been clearly demonstrated. Recently, we have shown that IpgD is one of the proteins potentially secreted by the S.flexneri type III secretion apparatus. IpgD is stored in the bacterial cytoplasm associated with a specific chaperone, IpgE. Although IpgD is not absolutely required for entry of bacteria into cultured cells (Allaoui et al., 1993), it is implicated in entry focus formation (Niebuhr et al., 2000). The sequence of IpgD exhibits two motifs that are present in the active site of mammalian inositol polyphosphate 4-phosphatase. Phosphoinositides, whose intracellular levels are controlled accurately by a complex set of kinases, phosphatases and phospholipases, play a key role in many processes including reorganization of the actin cytoskeleton (Toker, 1998; Czech, 2000; Sechi and Wehland, 2000; Payrastre et al., 2001) and plasma membrane–cytoskeleton linkage (Raucher et al., 2000). Here, we demonstrate that IpgD is translocated directly into the host cell, where it functions as a phosphoinositide phosphatase that dephosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to generate the novel lipid phosphatidylinositol 5-monophosphate [PtdIns(5)P]. Furthermore, we show that expression of IpgD in mammalian cells leads to a strong reduction in tether force that eventually causes membrane blebbing. Our results strongly suggest that IpgD uncouples the cellular plasma membrane from the actin cytoskeleton by locally reducing its adhesion energy through the transformation of PtdIns(4,5)P2 into PtdIns(5)P during S.flexneri entry. The system we describe is a novel strategy used by a pathogenic bacterium that dramatically and specifically disturbs a key element of phosphoinositide metabolism at the plasma membrane to increase its virulence. Results IpgD is a phosphoinositide phosphatase The sequence of IpgD contains two motifs related to the active site of mammalian inositol polyphosphate 4-phosphatases (Norris et al., 1998), and it has been shown that Salmonella dublin and S.typhimurium proteins homologous to IpgD, SopB and SigD, respectively, are endowed with an inositol phosphate phosphatase activity (Norris et al., 1998; Marcus et al., 2001). To test the activity and the substrate specificity of IpgD, we used a purified GST–IpgD fusion protein expressed together with the chaperone IpgE. We found that it hydrolysed several phosphoinositides in vitro (Table I) and exhibited the greatest activity towards PtdIns(4,5)P2. Very weak or no activity was detected towards the inositol phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This substrate specificity was not due to the presence of IpgE in the preparation of GST–IpgD since similar results were obtained when IpgD was produced without IpgE (data not shown). Replacement of the cysteine residue in the proposed catalytic domain of IpgD by a serine residue (C438S) led to a protein that had no activity against PtdIns(4,5)P2 (not shown) as classically observed with this phosphatase family (Norris et al., 1997). The substrate specificity of IpgD in vitro appears different from that of SopB or SigD, which have a preference for phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] (Norris et al., 1998). As previously noted for SopB (Norris et al., 1997), the specific activity of IpgD was relatively low compared with the activity of mammalian homologues, which may reflect the difficulty in setting appropriate conditions for in vitro lipid phosphatase assays for these proteins. Table 1. Phosphatase activity of recombinant IpgD Substrate nmol/min/mg protein PtdIns(4)P 10.0 ± 5 PtdIns(3)P 5.8 ± 3 PtdIns(4,5)P2 61.0 ± 9 PtdIns(3,4)P2 25.3 ± 2 PtdIns(3,4,5)P3 32.1 ± 10 Ins(1,4,5)P3 0.3 ± 0.1 Ins(1,3,4,5)P4 ND Assays were performed using exogenous lipids or inositol phosphates as indicated in Materials and methods. Results are the mean ± SEM from 3–4 independent experiments, except those for inositol phosphate phosphatase activity, which are from two experiments. ND = not detectable. IpgD hydrolyses PtdIns(4,5)P2 during infection of epithelial cells by S.flexneri To investigate the role of IpgD on the cellular levels and/or turnover of the various phosphoinositides during infection of HeLa cells, we analysed the phospholipid content of cells labelled with 32Pi and infected with the wild-type strain M90T, the ipgD mutant and the non-invasive virulence plasmid cured strain BS176, over time. As soon as 15 min after contact between bacteria and host cells, cellular [32P]PtdIns(4,5)P2 levels dropped dramatically with a concomitant increase in [32P]PtdInsP (Figure 1A and C). In contrast, the levels of [32P] PtdIns(4,5)P2 and [32P]PtdInsP in cells infected with the ipgD mutant or with the non-invasive strain did not change, even after 30 min of infection (Figure 1B and C), demonstrating the absolute requirement for IpgD in the hydrolysis of [32P]PtdIns(4,5)P2. The levels of the major cellular [32P]phospholipids (i.e. phosphatidylcholine, -serine and -ethanolamine) (Figure 1A) as well as [32P]phosphatidylinositol (PtdIns) (data not shown) and PtdIns(3)P (see Figure 4) were not affected, indicating that IpgD phosphatase activity was directed towards [32P]PtdIns(4,5)P2 in vivo. Thus, IpgD appears as a S.flexneri effector that affects the level of specific host cell polyphosphoinositides during infection. Figure 1.IpgD-dependent PtdIns(4,5)P2 hydrolysis in HeLa cells infected with S.flexneri. HeLa cells were labelled with [32P]orthophosphate and infected with either the wild-type M90T (WT) (A) or the ipgD (ipgD) (B) strains. After infection, cells were washed with PBS and reactions were stopped by adding ice-cold HCl (2.4 M). Cells were recovered by scraping, and lipids were extracted and separated by TLC (left panel). The radiolabelled PtdInsP + PtdInsP2 were recovered by scraping the appropriate bands as indicated, and were deacylated and analysed by HPLC (right panels). MP, major phospholipids; CTS, counts per second. (C) Quantification of [32P]PtdInsP [PtdIns(4)P + PtdIns(5)P which co-elute with the classical HPLC technique] (diamonds) and [32P]PtdIns(4,5)P2 (squares) in HeLa cells infected either with WT, ipgD or the avirulant, non-invasive mutant BS176. Data shown are representative of 4–6 independent experiments with similar results. Download figure Download PowerPoint Figure 2.Production of PtdIns(5)P without changes in PtdIns(4)P levels during Shigella infection. HeLa cells were labelled with [32P]orthophosphate and infected with either ipgD mutant (ipgD) (A) or wild-type M90T (WT) (B) strains for 30 min. Lipids were then extracted and analysed as described in Figure 1. Radiolabelled PtdInsP was recovered by scraping the corresponding band from the TLC plate as indicated, and was then deacylated and analysed by an appropriate HPLC technique (Tolias et al., 1998) allowing the separation of PtdIns(4)P and PtdIns(5)P (right panel). Peaks corresponding to these phosphoinositides are indicated. Data are from one experiment, representative of three. Download figure Download PowerPoint Figure 3.Quantification of IpgD-dependent production of PtdIns(5)P by mass assay. (A) The mass level of PtdIns(5)P was measured in non- infected cells (−) or in cells infected with either M90T, ipgD or the avirulant, non-invasive mutant BS176 for 20 min using a specific mass assay as previously described by Morris et al. (2000). Recombinant PIPkinase IIα was used in the presence of [γ-32P]ATP to phosphorylate PtdIns(5)P present in the PtdInsP fraction extracted from cells infected with the different strains. The PtdIns(5)P was transformed specifically to [32P]PtdIns(4,5)P2, which was quantified. A representative TLC illustrating the production of [32P]PtdIns(4,5)P2 from PtdIns(5)P present in the PtdInsP fraction extracted from cells infected with various strains is shown (top panel). Results are also expressed as pmol PtdIns(5)P/mg of HeLa cell proteins and are the mean ± SEM of three independent experiments (lower panel). (B) The mass level of PtdIns(5)P was measured in HeLa cells transfected with GFP (C) or GFP-tagged IpgD (IpgD) after 24 h. Results are representative of three independent experiments. Download figure Download PowerPoint PtdIns(5)P is the product of the IpgD-dependent PtdIns(4,5)P2 degradation in S.flexneri-infected cells The increase of [32P]PtdInsP that was detected in parallel with the decrease of [32P]PtdIns(4,5)P2 suggested that IpgD specifically dephosphorylated one of the two positions of the inositol moiety leading to accumulation of the hydrolysis product. To identify the product of the enzymatic reaction, we analysed the nature of the [32P]PtdInsP produced in infected cells using an appropriate high-performance liquid chromatography (HPLC) technique allowing the separation of PtdIns(4)P and PtdIns(5)P (Tolias et al., 1998; Sbrissa et al., 1999). During the course of infection, the [32P]PtdIns(4)P levels remained unchanged whereas a second peak at the expected position for [32P]PtdIns(5)P was clearly detected (Figure 2). This recently discovered phosphoinositide has been shown to be quantitatively minor in several models (Rameh et al., 1997; Tolias et al., 1998) and indeed was undetectable by HPLC in resting HeLa cells. To confirm that PtdIns(5)P was accumulating in infected cells and to quantify its mass level, we performed a recently described mass assay (Morris et al., 2000). Recombinant PtdInsP–kinase IIα, whose major substrate is PtdIns(5)P, was used in the presence of [γ-32P]ATP to phosphorylate the PtdInsP fraction extracted from cells infected with the various strains. The PtdIns(5)P present in this fraction was transformed specifically to [32P]PtdIns(4,5)P2, which was quantified. As shown in Figure 3A, this assay allowed us to demonstrate unambiguously that the mass level of PtdIns(5)P dramatically increased during infection with the wild-type strain M90T (from 18.6 ± 6.9 to 280.5 ± 78.3 pmol/mg of HeLa cell proteins). Conversely, small amounts of PtdIns(5)P were detected in non-infected cells or in cells infected with either the ipgD mutant or BS176. To demonstrate further that IpgD produces PtdIns(5)P in mammalian cells, we measured the level of this phosphoinositide in HeLa cells transiently transfected by a green fluorescent protein (GFP)-tagged IpgD construct. As shown in Figure 3B, expression of IpgD induced the production of a significant amount of PtdIns(5)P. Altogether, these data demonstrated that, in vivo, IpgD specifically acts as a PtdIns(4,5)P2 4-phosphatase and leads to accumulation of PtdIns(5)P in the host cell. During S.flexneri infection, we also noticed an increase in PtdIns(3,4)P2, phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] and PtdIns(3,4,5)P3, indicating a concomitant activation of a phosphatidylinositol 3-kinase (PI 3-kinase) (Figure 4). The increase in these phosphoinositides during infection highlights the in vivo specificity of this bacterial phosphatase towards cellular PtdIns(4,5)P2, which appears to be the only phosphoinositide to be hydrolysed. These results also indicate that the in vitro activity of IpgD towards PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Table I) does not appear to reflect its activity in infected cells. Experiments performed with the non-invasive mutant (BS176) or the ipgD mutant indicated that activation of the PI 3-kinase pathway was linked to invasion (Figure 4C), as was also suggested recently for Salmonella (Marcus et al., 2001). This activation of PI 3-kinase, however, was not required for S.flexneri entry since treatment of HeLa cells with the PI 3-kinase inhibitor wortmannin had no effect on the entry process (data not shown). Figure 4.PI 3-kinase is activated during Shigella infection. HeLa cells were labelled with [32P]orthophosphate and infected with either the ipgD (A) or the wild-type M90T (WT) (B) strains for 30 min. Lipids were then extracted and analysed as described in Figure 1. The radiolabelled PtdInsP2 [PtdIns(3,5)P2, PtdIns(3,4)P2 and PtdIns(4,5)P2] + PtdIns(3,4,5)P3 were recovered by scraping the appropriate bands from the TLC plate, and were deacylated and analysed by HPLC (left panel). Peaks corresponding to PtdIns(3,5)P2 (1), PtdIns(3,4)P2 (2), PtdIns(4,5)P2 (3), ATP (4) and PtdIns(3,4,5)P3 (5) are indicated. (C) Quantification of [32P]D3 phosphoinositides in cells infected with either wild-type M90T (squares), ipgD mutant (diamonds) or the non-invasive mutant BS176 (circles). Data are from one experiment, representative of four. Download figure Download PowerPoint IpgD is translocated into the eukaryotic cell by extracellular bacteria The rapid degradation of cellular PtdIns(4,5)P2 upon contact with S.flexneri suggested that this mechanism might be an early event during the invasion process, preceding the entry of bacteria into host cells. To determine whether IpgD was injected into the host cell cytoplasm by extracellular bacteria or whether it was secreted by bacteria that had already reached the cytoplasm, HeLa cells were treated with cytochalasin D to prevent the actin rearrangements required for bacterial uptake (Clerc and Sansonetti, 1987). During infection in the presence of 0.5 μg/ml cytochalasin D, the entry of S.flexneri was blocked by 100%, whereas an important production of PtdIns(5)P was still observed (201.8 ± 50 pmol/mg of HeLa cell proteins in 0.5 μg/ml cytochalasin D-treated cells versus 280.5 ± 78.3 in non-treated cells). Accordingly, a drop in cellular [32P]PtdIns(4,5)P2 was also detected (30 ± 9% decrease). These results demonstrated that IpgD is injected into the host cell by extracellular bacteria as a part of the initial step of the infection process. IpgD causes membrane blebbing and cell rounding when expressed in HeLa cells A comparison between the wild-type and the ipgD mutant strains showed that the mutant elicited entry structures with a highly altered morphology (Niebuhr et al., 2000). In contrast to the wild-type strain, which caused prominent cell surface and actin rearrangements around its attachment sites, the ipgD mutant induced dense, cup-like actin structures beneath adherent bacteria that resembled focal adhesion plaques. To examine the effect of IpgD on eukaryotic cells in the absence of other bacterial factors, we transiently expressed a myc-tagged IpgD in HeLa cells. Transfected cells were analysed by SDS–PAGE, and IpgD was detected with antibodies against the tag or the protein itself, to confirm that IpgD was expressed as a protein of the expected size (not shown). Moreover, after 24 h of expression of IpgD in HeLa cells, the production of PtdIns(5)P was clearly detected (20 pmol/mg protein in non-transfected versus 122 pmol/mg protein in transfected cells), indicating that the transfected phosphatase was active. Analysis of transfected cells by confocal microscopy revealed that expression of IpgD affected cell morphology. After a period of 8–24 h, HeLa cells started to form membrane blebs that protruded up to 15 μm above the cell surface, while the actin stress fibres began to disappear (Figure 5). Expression of the inactive myc-tagged IpgD-C438S did not induce these morphological changes (not shown). Furthermore, expression of GFP-tagged IpgD in NIH-3T3 cells also affected their morphology. Again, after 8–24 h, these cells formed membrane blebs and the actin stress fibres disappeared (Figure 5C and E). As a control, expression of the inactive GFP-tagged IpgD-C438S mutant in NIH-3T3 cells did not significantly affect their morphology (Figure 5D and F). Figure 5.Expression of IpgD causes formation of membrane blebs. Confocal laser scan analysis of the surface structures elicited on HeLa cells transfected with myc-tagged IpgD after 24 h. For immunofluorescence, transfected cells were visualized using an anti-myc antibody (red), and filamentous actin was stained using FITC-coupled phalloidin. The blebs observed on such cells protruded up to 15 μm above the cells. (A) The sum of all optical sections. (B) The corresponding side view (z-projection). Alternatively, GFP-tagged IpgD (C and E) and GFP-tagged IpgDC438S mutant (D and F) were transfected in NIH-3T3 cells. After 24 h, the filamentous actin was stained using rhodamine-coupled phalloidin (C and D) and the preparations were observed by fluorescence microscopy, using a Zeiss Axioskop microscope equipped with a 63× objective and a Princeton microMAX camera. The data shown are representative of four independent experiments with similar results. Download figure Download PowerPoint IpgD decreases cytoskeletal–membrane adhesion when expressed in NIH-3T3 cells Tether force measurement was coupled with fluorescence quantification in NIH-3T3 cells that had been transfected with 0.5 μg of a GFP-tagged IpgD construct. Experiments were performed 8 h post-transfection. As shown in Figure 6A and B, cells expressing increasing levels of GFP-tagged IpgD molecules have an inversely proportional decrease in tether force. Transfected NIH-3T3 cells exhibit dynamic lamellar structures similar to those of transfected HeLa cells at lower concentrations and relatively uniform fluorescence of the fusion protein, as seen in Figure 6C. A blebbing phenotype was observed at concentrations >2 × 105 molecules of GFP-tagged IpgD. After calibration of the laser tweezers (Kuo and Sheetz, 1993), >5 tether force measurements (i.e. the force needed to hold a tethered bead at a constant distance from the trap) were performed in the course of three experiments. As shown in Figure 6A and B, tether force was inversely proportional to the amount of GFP-tagged IpgD fusion expressed in the relevant cell, and a significant decrease in membrane tension was observed even with low levels of expression of GFP-tagged IpgD. Figure 6.Membrane cytoskeleton adhesion energy is decreased, as seen by tether force in cells expressing IpgD–GFP fusion protein. After calibration of the laser tweezers, the displacement of the bead from the centre of the trap was converted to tether force (i.e. the force needed to hold a tether at a constant length). (A and B) Increasing levels of IpgD–GFP create an inversely proportional decrease in tether force. An expression level of 3.5 × 105 molecules of IpgD decreases the force by half. (C) NIH-3T3 cells transfected with IpgD–GFP show relatively uniform fluorescence with random punctate spots. Hazy fluorescence is due to the high turnover rate of active lamellae. A standardized bead was used to quantitate the number of molecules of IpgD–GFP. Bar = 7.4 μm. Download figure Download PowerPoint IpgD enhances the effect of Cdc42 and Rac agonists on cytoskeletal remodelling Cdc42 and Rac are essential regulators governing production of the filopodial and lamellipodial structures that form the entry focus of Shigella. It was shown previously that these structures were much shorter during entry of a Shigella ipgD mutant (Niebuhr et al., 2000). As shown in Figure 7, expression of IpgD by transfected HEK cells amplified and altered the structure of the cytoskeletal projections induced by bradykinin, an agonist of Cdc42 (Kozma et al., 1995), and by epidermal growth factor (EGF), an agonist of Rac (Azuma et al., 1998). Figure 7 shows the 'hairless' aspect of HEK cells and the lack of a significant effect of GFP expression on these cells (Figure 7A), whereas expression of the GFP-tagged IpgD fusion altered cytoskeletal structures, leading to a significant rounding of the cells and a concentration of the actin cytoskeleton at the periphery of these rounded cells (Figure 7D, arrows). The duration of transfection was such that cells did not reach the blebbing stage. In the presence of bradykinin, activation of Cdc42 led to the formation of thin but numerous filopodial structures at the cellular surface (Figure 7B, arrows), whereas those with bradykinin-induced filopodia were significantly extended in the cells expressing the GFP-tagged IpgD construct (Figure 7C, arrows). In the presence of EGF, activation of Rac led to the formation of lamellipodial structures at the cell periphery (Figure 7C, arrows). These lamellipodia were not only extended, but also exhibited modified morphology producing gigantic extensions (Figure 7F, arrows). These results are very consistent with the small size of entry foci induced by the Shigella ipgD mutant (Niebuhr et al., 2000). Figure 7.IpgD enhances the effect of Cdc42 and Rac agonists on cytoskeletal remodelling. HEK cells were transfected with GFP (A–C) or GFP-tagged IpgD (D–F). The duration of transfection was such that cells did not reach the blebbing stage. Cells were then stimulated (B, C, E and F) or not (A and D) with 0.1 μg/ml bradykinin for 20 min (B and E) or 5 nM EGF for 5 min (C and F), and filamentous actin was stained using rhodamine-coupled phalloidin. The preparations were observed by confocal microscopy. Data shown are representative of three independent experiments. Download figure Download PowerPoint Discussion Seven different polyphosphoinositides have been identified so far, and most of them play central roles in the control of fundamental cell functions, such as spatio-temporal organization of key signalling pathways, reorganization of the actin cytoskeleton or intracellular membrane trafficking (Toker, 1998; Czech, 2000; Odorizzi et al., 2000). A complex set of phosphoinositide-metabolizing enzymes, such as kinases and phosphatases, accurately regulates the level of these quantitatively minor lipid molecules. These enzymes, which seem to be targeted specifically to various intracellul