Regulation of Salmonella-induced membrane ruffling by SipA differs in strains lacking other effectors

The Salmonella pathogenicity island 1 (SPI-1) type three secretion system (TTSS) is essential for Salmonella invasion of host cells through its triggering of actin-dependent membrane ruffles. The SPI-1 effectors SipA, SopE, SopE2 and SopB all have actin regulating activities and contribute to invasion. The precise role of actin regulation by SipA in Salmonella invasion remains controversial since divergent data have been presented regarding the relationship between SipA and membrane ruffling. We hypothesized that the contribution of SipA to membrane ruffling and invasion might vary between Salmonella strains. We compared the effects of SipA deletion on Salmonella enterica serovar Typhimurium ( S.  Typhimurium) strains that possess or lack SopE. Loss of SipA reduced invasion in the early stages of infection by SopE⁺ and SopE^- strains but the number of membrane ruffles elicited was unaffected. Salmonella strains lacking both SipA and SopE induced ruffles with very different morphology from those induced by wild-type strains or ones lacking single effectors, including the presence of highly dynamic finger-like protrusions and numerous filopodia. A similar phenotype was found for sipA ^- sopE ^-, sipA ^- sopE2 ^- and sipA ^- sopB ^- mutants. Thus, SipA plays a more prominent role in induction of invasion-competent membrane ruffles by Salmonella lacking a full complement of SPI-1 effectors.     

Molecular basis of Salmonella-induced enteritis

Salmonella pathogenesis is a complex and multifactorial phenomenon. Many genes required for full virulence in mice have been identified, but only a few of these have been shown to be necessary for the induction of enteritis. Likewise, at least some of the Salmonella virulence factors affecting enteritis do not appear to be required for infection of systemic sites in mice. This suggests that subsets of virulence genes influence distinct aspects of Salmonella pathogenesis. Recently, considerable progress has been made in characterizing the virulence mechanisms influencing enteritis caused by non-typhoid Salmonella spp. The Salmonella pathogenicity island-1-encoded type III secretion system mediates the translocation of secreted effector proteins into target epithelial cells. These effector proteins are key virulence factors required for Salmonella intestinal invasion and the induction of fluid secretion and inflammatory responses.     

Malaria parasites harness Rho GTPase signaling and host cell membrane ruffling for productive invasion of hepatocytes

1. Download : Download high-res image (185KB)
2. Download : Download full-size image

     

Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells

Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct 'leader' domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.     

Molecular dissection of host cell invasion by the apicomplexans: the glideosome

Gliding motility is an essential and fascinating apicomplexan-typical adaptation to an intracellular lifestyle. Apicomplexan parasites rely on gliding motility for their migration across biological barriers and for host cell invasion and egress. This unusual substratedependent mode of locomotion involves the concerted action of secretory adhesins, a myosin motor, factors regulating actin dynamics and proteases. During invasion, complexes of soluble and transmembrane micronemes proteins (MICs) and rhoptry neck proteins (RONs) are discharged to the apical pole of the parasite, some protein acts as adhesins and bind to host cell receptors whereas others are involved in the moving junction formation. These complexes redistribute towards the posterior pole of the parasite via a physical connection to the parasite actomyosin system and are eventually released from the parasite surface by the action of parasite proteases.     

The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling

This study was performed to elucidate the host cell scaffolding and signalling molecules that Campylobacter jejuni utilizes to invade epithelial cells. We hypothesized that the C. jejuni fibronectin‐binding proteins and secreted proteins are required for cell signalling and maximal invasion of host cells. C. jejuni binding to host cells via the CadF and FlpA fibronectin‐binding proteins activated the epidermal growth factor (EGF) pathway, as evidenced by inhibitor studies and immunoprecipitation coupled with immunoblot analysis using antibodies reactive against total and active EGF receptor. Inhibitor studies revealed maximal C. jejuni host cell invasion was dependent upon PI3‐Kinase, c‐Src and focal adhesion kinase (FAK), all of which are known to participate in cytoskeletal rearrangements. Knockdown of endogenous Dock180, which is a Rac1‐specific guanine nucleotide exchange factor, using siRNA revealed that C. jejuni invasion was significantly reduced compared with cells treated with scrambled siRNA. We further demonstrated that the C. jejuni Cia proteins are, in part, responsible for Rho GTPase Rac1 recruitment and activation, as judged by immunofluorescence microscopy and Rac1 activation. Based on these data, we present a model that illustrates that C. jejuni utilizes a coordinated mechanism involving both adhesins and secreted proteins to promote membrane ruffling and host cell invasion.     

SPIN90-IRSp53 complex participates in Rac-induced membrane ruffling

SPIN90 is a key regulator of actin cytoskeletal organization. Using the BioGRID^beta database (General Repository for Interaction Datasets), we identified IRSp53 as a binding partner of SPIN90, and confirmed the in vivo formation of a SPIN90-IRSp53 complex mediated through direct association of the proline-rich domain (PRD) of SPIN90 with the SH3 domain of IRSp53. SPIN90 and IRSp53 positively cooperated to mediate Rac activation, and co-expression of SPIN90 and IRSp53 in COS-7 cells led to the complex formation of SPIN90-IRSp53 in the leading edge of cells. PDGF treatment induced strong colocalization of SPIN90 and IRSp53 at membrane protrusions. Within such PDGF-induced protrusions, knockdown of SPIN90 protein using siRNA significantly reduced lamellipodia-like protrusions as well as localization of IRSp53 at those sites. Finally, competitive inhibition of SPIN90-IRSp53 binding by SPIN90 PRD dramatically reduced ruffle formation, further suggesting that SPIN90 plays a key role in the formation of the membrane protrusions associated with cell motility.     

Lamellipodium extension and membrane ruffling require different SNARE-mediated trafficking pathways

Background  

Intracellular membrane traffic is an essential component of the membrane remodeling that supports lamellipodium extension during cell adhesion. The membrane trafficking pathways that contribute to cell adhesion have not been fully elucidated, but recent studies have implicated SNARE proteins. Here, the functions of several SNAREs (SNAP23, VAMP3, VAMP4 and syntaxin13) are characterized during the processes of cell spreading and membrane ruffling.     

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

Background  

Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.     

Molecular dissection of the membrane aggregation mechanisms induced by monomeric annexin A2

Annexins are a multigene family of proteins involved in aggregation and fusion processes of biological membranes. One of its best-known members is annexin A2 (or p36), capable of binding to acidic phospholipids in a calcium-dependent manner, as occurs with other members of the same family. In its heterotetrameric form, especially with protein S100A10 (p11), annexin A2 has been involved as a determinant factor in innumerable biological processes like tumor development or anticoagulation. However, the subcellular coexistence of different pools of the protein, in which the monomeric form of annexin A2 is growing in functional relevance, is to date poorly described. In this work we present an exhaustive structural and functional characterization of monomeric human annexin A2 by using different recombinant mutants. The important role of the amphipathic N-terminal α-helix in membrane binding and aggregation has been analyzed. We have also studied the potential implication of lateral “antiparallel” protein dimers in membrane aggregation. In contrast to what was previously suggested, formation of these dimers negatively regulate aggregation. We have also confirmed the essential role of three lysine residues located in the convex surface of the molecule in calcium-free and calcium-dependent membrane binding and aggregation. Finally, we propose models for annexin A2-mediated vesicle aggregation mechanisms.     

Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor

One of the earliest effects of platelet-derived growth factor (PDGF) on human fibroblasts in culture is an induction of membrane ruffling. The morphology of the ruffles induced by PDGF is unique in that they form circular arrangements on the dorsal side of the cells. Here we report that the induction of circular ruffle arrangements is an effect specific for PDGF, dose-dependent and inhibitable by anti-PDGF antibodies. We have attempted to utilize this effect to design a rapid and sensitive bioassay for PDGF. The "membrane ruffling assay" is compared with other methods to measure PDGF and its specificity with regard to the different dimeric forms of PDGF is discussed. Introduction of Ca2+ into the cells via the Ca2+ ionophore A23187 or the addition of the tumor-promor 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a stimulator of protein kinase C, does not induce circular ruffle formations on human fibroblasts, neither does the addition of the combination of these two agents. However, addition of TPA almost completely inhibits PDGF-induced circular ruffle formations. Further, we find a shift in the time-course of the PDGF-induced circular ruffle formations by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases. This may indicate the involvement of protein phosphorylation in the regulation of PDGF-induced membrane ruffling.     

The small GTP-binding protein rac regulates growth factor-induced membrane ruffling.

The function of rac, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, rac1 rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rac protein, N17rac1. This suggests that endogenous rac proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both rac1 microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17rac1 we have shown that these growth factors act through rac to stimulate this rho-dependent response. We propose that rac and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.     

Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells

Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.     

Analysis of the mechanisms of Salmonella-induced actin assembly during invasion of host cells and intracellular replication

Salmonella enterica serovar Typhimurium (S. typhimurium) induces actin assembly both during invasion of host cells and during the course of intracellular bacterial replication. In this study, we investigated the involvement in these processes of host cell signalling pathways that are frequently utilized by bacterial pathogens to manipulate the eukaryotic actin cytoskeleton. We confirmed that Cdc42, Rac, and Arp3 are involved in S. typhimurium invasion of HeLa cells, and found that N-WASP and Scar/WAVE also play a role in this process. However, we found no evidence for the involvement of these proteins in actin assembly during intracellular replication. Cortactin was recruited by Salmonella during both invasion and intracellular replication. However, RNA interference directed against cortactin did not inhibit either invasion or intracellular actin assembly, although it resulted in increased cell spreading and a greater number of lamellipodia. We also found no role for either the GTPase dynamin or the formin family member mDia1 in actin assembly by intracellular bacteria. Collectively, these data provide evidence that signalling pathways leading to Arp2/3-dependent actin nucleation play an important role in S. typhimurium invasion, but are not involved in intracellular Salmonella-induced actin assembly, and suggest that actin assembly by intracellular S. typhimurium may proceed by a novel mechanism.     

Localized RhoA activation as a requirement for the induction of membrane ruffling

We examined the spatio-temporal activity of RhoA in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In HeLa cells migrating at a low cell density, RhoA was activated both at the contractile tail and at the leading edge. However, RhoA was activated only at the leading edge in MDCK cells migrating as a monolayer sheet. In growth factor-stimulated Cos1 and NIH3T3 cells, the activity of RhoA was greatly decreased at the plasma membrane, but remained high at the membrane ruffles in nascent lamellipodia. These observations are in agreement with the proposed role played by RhoA in stress fiber formation, but they also implicated RhoA in the regulation of membrane ruffling, the induction of which is a typical phenotype of activated Rac. In agreement with this view, dominant negative RhoA was found to inhibit membrane ruffling induced by active Rac. Furthermore, we found that Cdc42 activity was also required for high RhoA activity in membrane ruffles. Finally, we found that mDia1, but not ROCK, was stably associated with membrane ruffles. In conclusion, these results suggested that RhoA cooperates with Rac1 and Cdc42 to induce membrane ruffles via the recruitment of mDia.     

Promotion of Salmonella typhimurium adherence and membrane ruffling in MDCK epithelia by staurosporine

Abstract Infection of Madin-Darby canine kidney epithelial cell monolayers with Salmonella typhimurium SL1344 for 60 min results in widespread bacterial invasion which is associated with remodelling of the apical cell membrane to form 'membrane ruffles'. Treatment of Madin-Darby canine kidney cell monolayers with the protein kinase inhibitor staurosporine resulted in a 12-fold increase in the number of adhered bacteria without significantly affecting bacterial invasion. Staurosporine treatment also significantly increased both the number and size of membrane ruffles. As S. typhimurium adhere preferentially to these areas of membrane lacking microvilli, the increased extent of membrane ruffling may explain the increased bacterial adherence. These data provide evidence that the propagation of membrane ruffles during S. typhimurium infection is modulated by changes in the phosphorylation state of host proteins.     

Pathogenicity of Salmonella: SopE-mediated membrane ruffling is independent of inositol phosphate signals

Studies [Zhou, D., Chen, L.-M., Hernandez, L., Shears, S.B., and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248-259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen's ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis.     

Abba promotes PDGF-mediated membrane ruffling through activation of the small GTPase Rac1

Abba is a member of the I-BAR-domain protein family that is characterized by a convex-shaped membrane-binding motif. Overexpression of GFP-tagged Abba in murine fibroblasts potentiated PDGF-mediated formation of membrane ruffles and lamellipodia. Immunofluorescent microscopy and pull-down analysis revealed that GFP-Abba colocalized with an active form of Rac1 in the membrane ruffles and enhanced the Rac GTPase activity in response to PDGF stimulation. Further immunoprecipitation assays demonstrated that GFP-Abba bound to both wild-type and constitutively active Rac1 and that the binding to either of the Rac1 forms was significantly enhanced upon PDGF stimulation. On the other hand, an Abba mutant deficient in Rac1 binding failed to promote membrane ruffling and Rac1 activation in response to PDGF. However, the cells overexpressing a truncated mutant carrying the I-BAR domain alone displayed numerous filopodia-like microspikes in a manner independent of growth factors. Also, the Rac-binding activity of the mutant was not affected by PDGF treatment. Our data indicates that the interaction between full-length Abba and Rac1 is implicated in membrane deformation and subjected to a growth factor-mediated regulation through the C-terminal sequence.     

v-Crk induces Rac-dependent membrane ruffling and cell migration in CAS-deficient embryonic fibroblasts

Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.     

Identification of a novel Rac1-interacting protein involved in membrane ruffling.

The Rac GTP binding proteins are implicated in actin cytoskeleton-membrane interaction in mammalian cells. In fibroblast cells, Rac has been shown to mediate growth factor-induced polymerization of actin to form membrane ruffles and lamellipodia. We report here the isolation of a noval Rac1-interacting protein, POR1. POR1 binds directly to Rac1, and the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1, V12Rac1, in quiescent rat embryonic fibroblast REF52 cells. Furthermore, POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways.