Exploiting pathogenic Escherichia coli to model transmembrane receptor signalling

Many microbial pathogens manipulate the actin cytoskeleton of eukaryotic target cells to promote their internalization, intracellular motility and dissemination. Enteropathogenic and enterohaemorrhagic Escherichia coli, which both cause severe diarrhoeal disease, can adhere to mammalian intestinal cells and induce reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria. As pedestal assembly is triggered by E. coli virulence factors that mimic several host cell-signalling components, such as transmembrane receptors, their cognate ligands and cytoplasmic adaptor proteins, it can serve as a powerful model system to study eukaryotic transmembrane signalling. Here, we consider the impact of recent data on our understanding of both E. coli pathogenesis and cell biology, and the rich prospects for exploiting these bacterial factors as versatile tools to probe cellular signalling pathways.     

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.     

Responding to attraction: chemotaxis and chemotropism in Dictyostelium and yeast

Polarized growth in response to external signals is essential for both the internal organization of cells and generation of complex multicellular structures during development. Oriented growth or movement requires specific detection of an external cue, reorganization of the cytoskeleton and subsequent growth or movement. Genetic approaches in both the budding yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum have shed light on the molecular and cellular aspects of growth or movement towards an external signal. This review discusses the mechanisms and signalling pathways that enable yeast and Dictyostelium cells to translate external signals into directed growth and movement, respectively.     

A vaccinia virus-driven interplay between the MKK4/7-JNK1/2 pathway and cytoskeleton reorganization

Viral manipulation of transduction pathways associated with key cellular functions such as survival, response to microbial infection, and cytoskeleton reorganization can provide the supportive milieu for a productive infection. Here, we demonstrate that vaccinia virus (VACV) infection leads to activation of the stress-activated protein kinase (SAPK)/extracellular signal-regulated kinase (ERK) 4/7 (MKK4/7)-c-Jun N-terminal protein kinase 1/2 (JNK1/2) pathway; further, the stimulation of this pathway requires postpenetration, prereplicative events in the viral replication cycle. Although the formation of intracellular mature virus (IMV) was not affected in MKK4/7- or JNK1/2-knockout (KO) cells, we did note an accentuated deregulation of microtubule and actin network organization in infected JNK1/2-KO cells. This was followed by deregulated viral trafficking to the periphery and enhanced enveloped particle release. Furthermore, VACV infection induced alterations in the cell contractility and morphology, and cell migration was reduced in the JNK-KO cells. In addition, phosphorylation of proteins implicated with early cell contractility and cell migration, such as microtubule-associated protein 1B and paxillin, respectively, was not detected in the VACV-infected KO cells. In sum, our findings uncover a regulatory role played by the MKK4/7-JNK1/2 pathway in cytoskeleton reorganization during VACV infection.     

Cytoskeleton reorganization in influenza hemagglutinin-initiated syncytium formation

Little is known about the mechanisms of cell-cell fusion in development and diseases and, especially, about fusion stages downstream of an opening of nascent fusion pore(s). Earlier works on different cell-cell fusion reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it has remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion stages. Here we explore cellular reorganization associated with fusion pore expansion in syncytium formation using relatively simple experimental system. Fusion between murine embryonic fibroblasts NIH3T3-based cells is initiated on demand by well-characterized fusogen influenza virus hemagglutinin. We uncouple early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage and establish that the transition from local fusion to syncytium requires metabolic activity of living cells. Effective syncytium formation for cells with disorganized actin and microtubule cytoskeleton argues against hypothesis that cytoskeleton drives fusion expansion.     

Mechanisms of Salmonella entry into host cells

Salmonella enterica is an enteric bacterial pathogen that causes a variety of food and water-borne diseases ranging from gastroenteritis to typhoid fever. Ingested bacteria colonize the intestinal epithelium by triggering their own phagocytosis, using a sophisticated array of effector proteins that are injected into the host cell cytoplasm through a type III secretion apparatus. The synergistic action of these secreted effectors leads to a dramatic reorganization of the host actin cytoskeleton, resulting in vigorous membrane protrusion and the engulfment of attached bacteria. Analysis of these effector proteins and identification of their cellular targets has provided insight into the molecular mechanisms by which bacteria can subvert the host signalling and cytoskeletal machinery for their own purposes. This review is intended to summarize our current understanding of the tools used by Salmonella to enter host cells, with a focus on effectors that modulate the actin cytoskeleton.     

Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, while the responses of Schwann cells during adhesion and migration are unknown, so we examined the expression changes of SSeCKS and F-actin in Schwann cells after exposure to fibronectin. Src (sarcoma) suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after Schwann cells adhesion and that SSeCKS increased during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we showed that Schwann cells in which SSeCKS expression was inhibited reduced cellular adhesion, spreading and promoted cellular migration on fibronectin through reorganization of actin stress fibers and blocking formation of focal adhesions. These results demonstrated SSeCKS modulate Schwann cells adhesion, spreading and migration by reorganization of the actin cytoskeleton.     

Tec kinases: shaping T-cell activation through actin

Following stimulation, T cells undergo marked actin-dependent changes in shape that are required for productive cellular interactions and movement during immune responses. Reorganization of the actin cytoskeletal is also necessary for the formation of an immunological synapse - the convergence of several signaling molecules at the plasma membrane that occurs after effective T-cell receptor (TCR) signaling. Much emerging evidence indicates that the Tec family of tyrosine kinases has a role in actin cytoskeleton reorganization. Specifically, T cells that lack or express mutant versions of the Tec kinase Itk show impaired TCR-induced actin polymerization, cell polarization and regulation of the signaling events involved in cytoskeletal reorganization. These data, as well as other findings, support roles for Tec kinases in actin cytoskeleton regulation.     

Filamin structure,function and mechanics: are altered filamin-mediated force responses associated with human disease?

The cytoskeleton framework is essential not only for cell structure and stability but also for dynamic processes such as cell migration, division and differentiation. The F-actin cytoskeleton is mechanically stabilised and regulated by various actin-binding proteins, one family of which are the filamins that cross-link F-actin into networks that greatly alter the elastic properties of the cytoskeleton. Filamins also interact with cell membrane-associated extracellular matrix receptors and intracellular signalling proteins providing a potential mechanism for cells to sense their external environment by linking these signalling systems. The stiffness of the external matrix to which cells are attached is an important environmental variable for cellular behaviour. In order for a cell to probe matrix stiffness, a mechanosensing mechanism functioning via alteration of protein structure and/or binding events in response to external tension is required. Current structural, mechanical, biochemical and human disease-associated evidence suggests filamins are good candidates for a role in mechanosensing.     

Signal transduction of phagocytosis

The interaction of particles with certain cell surface receptors initiates intracellular signalling pathways that ultimately lead to submembranous actin filament assembly, pseudopod extension, and the ingestion of the particles. Here, Steven Greenberg reviews recent evidence implicating various signalling events in phagocytosis--in particular, activation of tyrosine kinases and phosphatidylinositol 3-kinase--and speculates how they might regulate the actin cytoskeleton.     

Actin cytoskeleton dynamics and the cell division cycle

The network of actin filaments is one of the crucial cytoskeletal structures contributing to the morphological framework of a cell and which participates in the dynamic regulation of cellular functions. In adherent cell types, cells adhere to the substratum during interphase and spread to assume their characteristic shape supported by the actin cytoskeleton. This actin cytoskeleton is reorganized during mitosis to form rounded cells with increased cortical rigidity. The actin cytoskeleton is re-established after mitosis, allowing cells to regain their extended shape and attachment to the substratum. The modulation of such drastic changes in cell shape in coordination with cell cycle progression suggests a tight regulatory interaction between cytoskeleton signalling, cell–cell/cell–matrix adhesions and mitotic events. Here, we review the contribution of the actin cytoskeleton to cell cycle progression with an emphasis on the effectors responsible for the regulation of the actin cytoskeleton and integration of their activities with the cell cycle machinery.     

Rho GTPase signalling pathways in the morphological changes associated with apoptosis

The killing and removal of superfluous cells is an important step during embryonic development, tissue homeostasis, wound repair and the resolution of inflammation. A specific sequence of biochemical events leads to a form of cell death termed apoptosis, and ultimately to the disassembly of the dead cell for phagocytosis. Dynamic rearrangements of the actin cytoskeleton are central to the morphological changes observed both in apoptosis and phagocytosis. Recent research has highlighted the importance of Rho GTPase signalling pathways to these changes in cellular architecture. In this review, we will discuss how these signal transduction pathways affect the structure of the actin cytoskeleton and allow for the efficient clearance of apoptotic cells.     

Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture

Vaccinia virus (VACV), the model poxvirus, produces two types of infectious particles: mature virions (MVs) and extracellular virions (EVs). EV particles possess two membranes and therefore require an unusual cellular entry mechanism. By a combination of fluorescence and electron microscopy as well as flow cytometry, we investigated the cellular processes that EVs required to infect HeLa cells. We found that EV particles were endocytosed, and that internalization and infection depended on actin rearrangements, activity of Na(+)/H(+) exchangers, and signalling events typical for the macropinocytic mechanism of endocytosis. To promote their internalization, EVs were capable of actively triggering macropinocytosis. EV infection also required vacuolar acidification, and acid exposure in endocytic vacuoles was needed to disrupt the outer EV membrane. Once exposed, the underlying MV-like particle presumably fused its single membrane with the limiting vacuolar membrane. Release of the viral core into the host cell cytosol allowed for productive infection.     

Syntrophins entangled in cytoskeletal meshwork: Helping to hold it all together

Syntrophins are a family of 59 kDa peripheral membrane‐associated adapter proteins, containing multiple protein‐protein and protein‐lipid interaction domains. The syntrophin family consists of five isoforms that exhibit specific tissue distribution, distinct sub‐cellular localization and unique expression patterns implying their diverse functional roles. These syntrophin isoforms form multiple functional protein complexes and ensure proper localization of signalling proteins and their binding partners to specific membrane domains and provide appropriate spatiotemporal regulation of signalling pathways. Syntrophins consist of two PH domains, a PDZ domain and a conserved SU domain. The PH1 domain is split by the PDZ domain. The PH2 and the SU domain are involved in the interaction between syntrophin and the dystrophin‐glycoprotein complex (DGC). Syntrophins recruit various signalling proteins to DGC and link extracellular matrix to internal signalling apparatus via DGC. The different domains of the syntrophin isoforms are responsible for modulation of cytoskeleton. Syntrophins associate with cytoskeletal proteins and lead to various cellular responses by modulating the cytoskeleton. Syntrophins are involved in many physiological processes which involve cytoskeletal reorganization like insulin secretion, blood pressure regulation, myogenesis, cell migration, formation and retraction of focal adhesions. Syntrophins have been implicated in various pathologies like Alzheimer’s disease, muscular dystrophy, cancer. Their role in cytoskeletal organization and modulation makes them perfect candidates for further studies in various cancers and other ailments that involve cytoskeletal modulation. The role of syntrophins in cytoskeletal organization and modulation has not yet been comprehensively reviewed till now. This review focuses on syntrophins and highlights their role in cytoskeletal organization, modulation and dynamics via its involvement in different cell signalling networks.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

How cells integrate complex stimuli: the effect of feedback from phosphoinositides and cell shape on cell polarization and motility

To regulate shape changes, motility and chemotaxis in eukaryotic cells, signal transduction pathways channel extracellular stimuli to the reorganization of the actin cytoskeleton. The complexity of such networks makes it difficult to understand the roles of individual components, let alone their interactions and multiple feedbacks within a given layer and between layers of signalling. Even more challenging is the question of if and how the shape of the cell affects and is affected by this internal spatiotemporal reorganization. Here we build on our previous 2D cell motility model where signalling from the Rho family GTPases (Cdc42, Rac, and Rho) was shown to organize the cell polarization, actin reorganization, shape change, and motility in simple gradients. We extend this work in two ways: First, we investigate the effects of the feedback between the phosphoinositides (PIs) , and Rho family GTPases. We show how that feedback increases heights and breadths of zones of Cdc42 activity, facilitating global communication between competing cell “fronts”. This hastens the commitment to a single lamellipodium initiated in response to multiple, complex, or rapidly changing stimuli. Second, we show how cell shape feeds back on internal distribution of GTPases. Constraints on chemical isocline curvature imposed by boundary conditions results in the fact that dynamic cell shape leads to faster biochemical redistribution when the cell is repolarized. Cells with frozen cytoskeleton, and static shapes, consequently respond more slowly to reorienting stimuli than cells with dynamic shape changes, the degree of the shape-induced effects being proportional to the extent of cell deformation. We explain these concepts in the context of several in silico experiments using our 2D computational cell model.     

Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 alter mitochondrial morphology during lytic replication

Epstein-Barr virus (EBV) is a human DNA virus that is responsible for the syndrome infectious mononucleosis, and is associated with several forms of cancer. During both lytic and latent viral infection, viral proteins manipulate the host's cellular components to aid in viral replication and maintenance. Here, it is demonstrated that induction of EBV lytic replication results in a dramatic reorganization of mitochondria accompanied by a significant alteration of mitochondrial membrane potential and a rapid and transient increase in the microtubular cytoskeleton. Moreover, we show that expression of the EBV immediate-early genes BZLF1 and BRLF1 contributes to the mitochondrial alteration but not the increase in the microtubule cytoskeleton, suggesting that the mechanism for the observed cytoplasmic restructuring involves a number of coordinated viral and host proteins.