A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.     

Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures

The bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate.     

αII-Spectrin Regulates Invadosome Stability and Extracellular Matrix Degradation

Invadosomes are actin-rich adhesion structures involved in tissue invasion and extracellular matrix (ECM) remodelling. αII-Spectrin, an ubiquitous scaffolding component of the membrane skeleton and a partner of actin regulators (ABI1, VASP and WASL), accumulates highly and specifically in the invadosomes of multiple cell types, such as mouse embryonic fibroblasts (MEFs) expressing SrcY527F, the constitutively active form of Src or activated HMEC-1 endothelial cells. FRAP and live-imaging analysis revealed that αII-spectrin is a highly dynamic component of invadosomes as actin present in the structures core. Knockdown of αII-spectrin expression destabilizes invadosomes and reduces the ability of the remaining invadosomes to digest the ECM and to promote invasion. The ECM degradation defect observed in spectrin-depleted-cells is associated with highly dynamic and unstable invadosome rings. Moreover, FRAP measurement showed the specific involvement of αII-spectrin in the regulation of the mobile/immobile β3-integrin ratio in invadosomes. Our findings suggest that spectrin could regulate invadosome function and maturation by modulating integrin mobility in the membrane, allowing the normal processes of adhesion, invasion and matrix degradation. Altogether, these data highlight a new function for spectrins in the stability of invadosomes and the coupling between actin regulation and ECM degradation.     

Actin dynamics at sites of extracellular matrix degradation

The degradation of extracellular matrix (ECM) by proteases is crucial in physiological and pathological cell invasion alike. In vitro, degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Here we present an extensive morpho-functional analysis of invadopodia actively engaged in ECM degradation and show that they are actin comet-based structures, not unlike the well-known bacteria-propelling actin tails. The relative mapping of the basic molecular components of invadopodia to actin tails is also provided. Finally, a live-imaging analysis of invadopodia highlights the intrinsic long-term stability of the structures coupled to a highly dynamic actin turnover. The results offer new insight into the tight coordination between signalling, actin remodelling and trafficking activities occurring at sites of focalized ECM degradation by invadopodia. In conclusion, invadopodia-associated actin comets are a striking example of consistently arising, spontaneous expression of actin-driven propulsion events that also represent a valuable experimental paradigm.     

Cortactin branches out: roles in regulating protrusive actin dynamics

Since its discovery in the early 1990's, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures.     

Palladin regulation of the actin structures needed for cancer invasion

Cell migration and invasion involve the formation of cell adhesion structures as well as the dynamic and spatial regulation of the cytoskeleton. The adhesive structures known as podosomes and invadopodia share a common role in cell motility, adhesion, and invasion, and form when the plasma membrane of motile cells undergoes highly regulated protrusions. Palladin, a molecular scaffold, co-localizes with actin-rich structures where it plays a role in their assembly and maintenance in a wide variety of cell lines. Palladin regulates actin cytoskeleton organization as well as cell adhesion formation. Moreover, palladin contributes to the invasive nature of cancer metastatic cells by regulating invadopodia formation. Palladin seems to regulate podosome and invodopodia formation through Rho GTPases, which are known as key players in coordinating the cellular responses required for cell migration and metastasis.     

Palladin regulation of the actin structures needed for cancer invasion

Cell migration and invasion involve the formation of cell adhesion structures as well as the dynamic and spatial regulation of the cytoskeleton. The adhesive structures known as podosomes and invadopodia share a common role in cell motility, adhesion, and invasion, and form when the plasma membrane of motile cells undergoes highly regulated protrusions. Palladin, a molecular scaffold, co-localizes with actin-rich structures where it plays a role in their assembly and maintenance in a wide variety of cell lines. Palladin regulates actin cytoskeleton organization as well as cell adhesion formation. Moreover, palladin contributes to the invasive nature of cancer metastatic cells by regulating invadopodia formation. Palladin seems to regulate podosome and invodopodia formation through Rho GTPases, which are known as key players in coordinating the cellular responses required for cell migration and metastasis.     

Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection

Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P(2)) and trisphosphate (PI[3,4,5]P(3)) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5'-phosphatase OCRL dephosphorylates PI(4,5)P(2) and to a lesser extent PI(3,4,5)P(3), we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P(2), and PI(3,4,5)P(3) levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating actin dynamics at bacterial internalization sites.     

The ubiquitin C‐terminal hydrolase UCH‐L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C‐terminal hydrolase UCH‐L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH‐L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH‐L1‐negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH‐L1 controls the early membrane‐associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c‐cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2‐ and AKT‐dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH‐L1_C90S. These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH‐L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria‐based drug delivery systems.     

Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection

Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans -membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott–Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253–276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1–21 are necessary and sufficient for its translocation, while amino acids 1–181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.     

Talin regulates moesin–NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin–NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.     

Repeated cycles of rapid actin assembly and disassembly on epithelial cell phagosomes

We have found that early in infection of the intracellular pathogen Listeria monocytogenes in Madin-Darby canine kidney epithelial cells expressing actin conjugated to green fluorescent protein, F-actin rapidly assembles (approximately 25 s) and disassembles (approximately 30 s) around the bacteria, a phenomenon we call flashing. L. monocytogenes strains unable to perform actin-based motility or unable to escape the phagosome were capable of flashing, suggesting that the actin assembly occurs on the phagosome membrane. Cycles of actin assembly and disassembly could occur repeatedly on the same phagosome. Indirect immunofluorescence showed that most bacteria were fully internalized when flashing occurred, suggesting that actin flashing does not represent phagocytosis. Escherichia coli expressing invA, a gene product from Yersinia pseudotuberculosis that mediates cellular invasion, also induced flashing. Furthermore, polystyrene beads coated with E-cadherin or transferrin also induced flashing after internalization. This suggests that flashing occurs downstream of several distinct molecular entry mechanisms and may be a general consequence of internalization of large objects by epithelial cells.     

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.     

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.     

Emerging roles of Abl family tyrosine kinases in microbial pathogenesis

Abl family kinases are central regulators of multiple cellular processes controlling actin dynamics, proliferation and differentiation. Recent studies indicate that different pathogens highjack Abl kinase signalling to reorganize the host actin cytoskeleton and promote the tyrosine phosphorylation of four known bacterial and viral effector proteins. Abl signalling is implicated in such diverse processes as microbial invasion, viral release from host cells, actin-based motility, actin-rich pedestal formation and cell scattering. Thus, Abl kinases are emerging as crucial regulators of multiple pathological signalling cascades during infection. Therapeutic intervention against Abl kinase activity might be an effective and novel strategy to combat serious microbial diseases.     

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus‐mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high‐resolution quantitative live‐cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili‐associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl‐β‐cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria‐induced plasma membrane reorganization is a rapid event driven by a direct cross‐talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.     

Spatial regulation of tumor cell protrusions by RhoC

Systemic metastasis is the dissemination of cancer cells from the primary tumor to distant organs and is the primary cause of death in cancer patients. How do cancer cells leave the primary tumor mass? The ability of the tumor cells to form different types of actin-rich protrusions including invasive protrusions (invadopodia) and locomotory protrusions (lamellipodia [2D] or pseudopodia [3D]), facilitate the invasion and dissemination of the tumor cells. Rho-family of p21 small GTPases plays a direct role in regulating the actin dynamics in these intracellular compartments. Recent studies have shown that the signaling molecules including RhoC/p190RhoGEF/p190RhoGAP acts as a “molecular compass” in order to direct the spatial and temporal dynamics of the formation of these invasive and locomotory protrusions leading to efficient invasion.     

Podosomes: adhesion hot-spots of invasive cells

Podosomes are highly dynamic, actin-rich adhesion structures of monocyte-derived cells, certain transformed fibroblasts and carcinoma cells and have recently also been discovered in an increasing number of other cell types. Because they are found mainly in motile cells and control the activity of matrix metalloproteases, podosomes are thought to contribute to tissue invasion and matrix remodeling. Importantly, podosomes are physiologically relevant organelles because they can be found in ex vivo models of invasive cells. Regulators of podosome turnover include tyrosine kinases, RhoGTPases, actin regulators and the microtubule system. Podosomes might also serve as an attractive model to study how integration of various signaling pathways controls actin dynamics. Here, we summarize and discuss the known structural, regulatory and functional features of podosomes, our aim being to stimulate further research into these unique structures.     

Involvement of SipA in modulating actin dynamics during Salmonella invasion into cultured epithelial cells

Salmonella entry into epithelial host cells results from the host actin cytoskeleton reorganization that is induced by a group of bacterial proteins delivered to the host cells by the Salmonella type III secretion system. SopE, SopE2 and SopB activate CDC42 and Rac1 to intercept the signal transduction pathways involved in actin cytoskeleton rearrangements. SipA and SipC directly bind actin to modulate the actin dynamics facilitating bacterial entry. Biochemical studies have indicated that SipA decreases the critical concentration for actin polymerization and may be involved in promoting the initial actin polymerization in Salmonella-induced actin reorganization. In this report, we conducted experiments to analyze the in vivo function(s) of SipA during Salmonella invasion. SipA was found to be preferentially associated with peripheral cortical actin filaments but not stress fibres using permeabilized epithelial cells. When polarized Caco-2 cells were infected with Salmonella, actin cytoskeleton rearrangements induced by the wild-type strain had many filopodia structures that were intimately associated with the bacteria. In contrast, ruffles induced by the sipA null mutant were smoother and distant from the bacteria. We also found that the F-actin content in cells infected with the sipA mutant decreased nearly 80% as compared to uninfected cells or those infected with the wild-type Salmonella strain. Furthermore, expression of either the full-length or the SipA(459-684) actin-binding fragment induced prominent punctuate actin assembly in the cortical region of COS-1 cells. These results indicate that SipA is involved in modulating actin dynamics in cultured epithelial cells during Salmonella invasion.     

Ezrin and EBP50 redistribute apically in rat uterine epithelial cells at the time of implantation and in response to cell contact

Uterine epithelial cells (UECs) undergo extensive morphological remodelling in preparation for an implanting blastocyst. This remodelling involves changes in the actin cytoskeleton and surface structures including microvilli. Ezrin and ezrin-radixin-moesin-binding protein-50-kDa (EBP50) link actin filaments to intra-membranous adhesion molecules and are important molecules in polarised epithelia. The current study is the first to describe the colocalisation and molecular association of ezrin and EBP50 in rat UECs by using immunofluorescence microscopy and immunoprecipitation techniques. These proteins have also been localised in relation to uterine epithelial cytoskeletal rearrangement during early pregnancy in the rat and to the effect of apical surface contact between opposing epithelial cells, blastocyst contact and contact with a silicon filament. Immunofluorescence microscopy has revealed that ezrin and EBP50 respond to contact between opposing epithelial cells and increase apically on day 6 of pregnancy. This apical distribution is also observed in UECs in contact with a silicon filament. Ezrin and EBP50 are however absent within the implantation chamber itself, seemingly mimicking the events that take place in leucocyte-endothelium binding. Thus, ezrin and EBP50 occur apically in UECs at the time of implantation in the rat and in response to a substitute blastocyst (filament) suggesting a role for these proteins in the cytoskeletal rearrangements that facilitate uterine receptivity and blastocyst-epithelial adhesion. Their loss within the implantation chamber possibly allows the subsequent invasion of the embryo.