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Actin Polymerization (actin + polymerization)
Selected AbstractsAfrican swine fever virus induces filopodia-like projections at the plasma membraneCELLULAR MICROBIOLOGY, Issue 11 2006Nolwenn Jouvenet Summary When exiting the cell vaccinia virus induces actin polymerization and formation of a characteristic actin tail on the cytosolic face of the plasma membrane, directly beneath the extracellular particle. The actin tail acts to propel the virus away from the cell surface to enhance its cell -to-cell spread. We now demonstrate that African swine fever virus (ASFV), a member of the Asfarviridae family, also stimulates the polymerization of actin at the cell surface. Intracellular ASFV particles project out at the tip of long filopodia-like protrusions, at an average rate of 1.8 µm min,1. Actin was arranged in long unbranched parallel arrays inside these virus-tipped projections. In contrast to vaccinia, this outward movement did not involve recruitment of Grb2, Nck1 or N-WASP. Actin polymerization was not nucleated by virus particles in transit to the cell periphery, and projections were not produced when the secretory pathway was disrupted by brefeldin A treatment. Our results show that when ASFV particles reach the plasma membrane they induce a localized nucleation of actin, and that this process requires interaction with virus-encoded and/or host proteins at the plasma membrane. We suggest that ASFV represents a valuable new model for studying pathways that regulate the formation of filopodia. [source] Plectin deposition at podosome rings requires myosin contractilityCYTOSKELETON, Issue 8 2008Annica Gad Abstract Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neuronsCYTOSKELETON, Issue 5 2007Daisuke Takahashi Abstract Axons and dendrites of neurons differ in the polarity orientation of their microtubules. Whereas the polarity orientation of microtubules in axons is uniform, with all plus ends distal, that in dendrites is nonuniform. The mechanisms responsible for establishment and maintenance of microtubule polarity orientation in neuronal processes remain unclear, however. We previously described a culture system in which dendrites of rat cortical neurons convert to axons. In the present study, we examined changes in microtubule polarity orientation in such dendrites. With the use of the hooking procedure and electron microscopy, we found that microtubule polarity orientation changed from nonuniform to uniform, with a plus end-distal arrangement, in dendrites that gave rise to axons during culture of neurons for 24 h. Microtubule polarity orientation remained nonuniform in dendrites that did not elongate. Axon regeneration at the dendritic tip thus triggered the disappearance of minus end-distal microtubules from dendrites. These minus end-distal microtubules also disappeared from dendrites during axon regeneration in the presence of inhibitors of actin polymerization, suggesting that actin-dependent transport of microtubules is not required for this process and implicating a previously unidentified mechanism in the establishment and maintenance of microtubule polarity orientation in neuronal processes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,CYTOSKELETON, Issue 4 2004Mike Lorenz Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. © 2004 Wiley-Liss, Inc. [source] Quantification of Shigella IcsA required for bacterial actin polymerizationCYTOSKELETON, Issue 4 2002Juana Magdalena Abstract Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500,2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella -infected cells. Cell Motil. Cytoskeleton 51:187,196, 2002. © 2002 Wiley-Liss, Inc. [source] Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1DEVELOPMENTAL NEUROBIOLOGY, Issue 8 2010Bonnie M. Marsick Abstract Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here, we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 565,588, 2010 [source] HGF induction of postsynaptic specializations at the neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2006Raghavan Madhavan Abstract A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Expression of PI(4,5)P2 -binding proteins lowers the PI(4,5)P2 level and inhibits Fc,RIIA-mediated cell spreading and phagocytosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Ewelina Szyma Abstract We found that Fc,RII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase I, (PIP5-kinase I,) that colocalized with PI(4,5)P2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C374,440 fragment of PIP5-kinase I, or the pleckstrin homology domain of phospholipase C,1 (PLC,1 -PH), two probes binding PI(4,5)P2. These probes reduced the amount of PI(4,5)P2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P2 elevation during phagocytosis. Simultaneously, PLC,1 -PH-GFP reduced the amount of PIP5-kinase I, associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase I,-GST bound PI(4,5)P2, phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLC,1 -PH domain, and possibly also the C374,440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase I, for PI(4,5)P2 binding in the plasma membrane leading eventually to PI(4,5)P2 depletion. [source] The actin-binding protein profilin I is localized at synaptic sites in an activity-regulated mannerEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005Henrike Neuhoff Abstract Morphological changes at synaptic specializations have been implicated in regulating synaptic strength. Actin turnover at dendritic spines is regulated by neuronal activity and contributes to spine size, shape and motility. The reorganization of actin filaments requires profilins, which stimulate actin polymerization. Neurons express two independent gene products , profilin I and profilin II. A role for profilin II in activity-dependent mechanisms at spine synapses has recently been described. Although profilin I interacts with synaptic proteins, little is known about its cellular and subcellular localization in neurons. Here, we investigated the subcellular distribution of this protein in brain neurons as well as in hippocampal cultures. Our results indicate that the expression of profilin I varies in different brain regions. Thus, in cerebral cortex and hippocampus profilin I immunostaining was associated predominantly with dendrites and was present in a subset of dendritic spines. In contrast, profilin I in cerebellum was associated primarily with presynaptic structures. Profilin I immunoreactivity was partially colocalized with the synaptic molecules synaptophysin, PSD-95 and gephyrin in cultured hippocampal neurons, indicating that profilin I is present in only a subset of synapses. At dendritic spine structures, profilin I was found primarily in protrusions, which were in apposition to presynaptic terminal boutons. Remarkably, depolarization with KCl caused a moderate but significant increase in the number of synapses containing profilin I. These results show that profilin I can be present at both pre- and postsynaptic sites and suggest a role for this actin-binding protein in activity-dependent remodelling of synaptic structure. [source] The first CH domain of affixin activates Cdc42 and Rac1 through ,PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factorGENES TO CELLS, Issue 3 2004Wataru Mishima Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with ,PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of ,PIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of ,PIX, ,PIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation. [source] Vacuolar membrane dynamics revealed by GFP-AtVam3 fusion proteinGENES TO CELLS, Issue 7 2002Tomohiro Uemura Background: The plant vacuole is a multifunctional organelle that has various physiological functions. The vacuole dynamically changes its function and shape, dependent on developmental and physiological conditions. Our current understanding of the dynamic processes of vacuolar morphogenesis has suffered from the lack of a marker for observing these processes in living cells. Results: We have developed transgenic Arabidopsis thaliana expressing a vacuolar syntaxin-related molecule (AtVam3/SYP22) fused with green fluorescent protein (GFP). Observations using confocal laser scanning microscopy demonstrated that the plant vacuole contained a dynamic membrane system that underwent a complex architectural remodelling. Three-dimensional reconstitution and time-lapse analysis of GFP-fluorescence images revealed that cylindrical and sheet-like structures were present in the vacuolar lumen and were moving dynamically. The movement, but not the structure itself, was abolished by cytochalasin D, an inhibitor of actin polymerization. This moving structure, which sometimes penetrated through the vacuolar lumen, possessed a dynamic membrane architecture similar to the previously recognized ,transvacuolar strand.' Conclusion: We propose two possible models for the formation of the vacuolar lumenal structure. Membrane structures including protruding tubules and reticular networks have recently been recognized in many other organelles, and may be actively involved in intra- and/or inter-organelle signalling. [source] HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasionGLIA, Issue 10 2008William A. Golembieski Abstract Secreted protein acidic and rich in cysteine (SPARC) regulates cell,extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC-green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC-induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC-regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC-induced glioma invasion. © 2008 Wiley-Liss, Inc. [source] IFN-, withdrawal after immunotherapy potentiates B16 melanoma invasion and metastasis by intensifying tumor integrin ,v,3 signalingINTERNATIONAL JOURNAL OF CANCER, Issue 3 2008Wei Gong Abstract Immunotherapy can effectively suppress tumor, yet complete tumor eradication occurs infrequently. The metastatic potential of remnant tumor cells after immunotherapy and the underlying mechanisms have not been fully elucidated. Here, we report that the termination of immunotherapy strikingly increases the metastatic potential of remnant melanoma. This is mainly due to the withdrawal of IFN-, after immunotherapy. The relief of IFN-, stress led to the increase of ,v,3 integrin expression in B16 cells, which increased the adhesion of B16 cells to fibrinogen, fibronectin and laminin. Through ,v,3 signaling, the activation of FAK, upregulation of cdc2, production of active MMP-2 and MMP-9 and actin polymerization were intensified in B16 cells stimulated with ECM molecules 24 h after the withdrawal of IFN-,. The i.v. injection of such tumor cells into mice resulted in more metastatic tumor nodes in lung and shortened the survival of mice. The pitfall of immunotherapy termination can be remedied by the administration of recombinant CBD-HepII polypeptide of fibronectin, which effectively inhibits ,v,3 signaling. These findings suggest that the risk of tumor metastasis can be increased after the termination of immunotherapy, due to the withdrawal of IFN-, and that targeting ,v,3 signaling pathway can improve the therapeutic effect of immunotherapeutic approaches by reducing such metastatic risk. © 2008 Wiley-Liss, Inc. [source] The cofilin activity cycle in lamellipodia and invadopodiaJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Matthew Oser Abstract The actin severing protein cofilin is essential for directed cell migration and chemotaxis, in many cell types and is also important for tumor cell invasion during metastasis. Through its severing activity, cofilin increases the number of free barbed ends to initiate actin polymerization for actin-based protrusion in two distinct subcellular compartments in invasive tumor cells: lamellipodia and invadopodia. Cofilin severing activity is tightly regulated and multiple mechanisms are utilized to regulate cofilin activity. In this prospect, we have grouped the primary on/off regulation into two broad categories, both of which are important for inhibiting cofilin from binding to F-actin or G-actin: (1) Blocking cofilin activity by the binding of cofilin to either PI(4,5)P2 at lamellipodia, or cortactin at invadopodia. (2) Blocking cofilin's ability to bind to actin via serine phosphorylation. Although the literature suggests that these cofilin regulatory mechanisms may be cell-type dependent, we propose the existence of a common cofilin activity cycle in which both operate. In this common cycle, the mechanism used to initiate cofilin activity is determined by the starting point in the cycle in a given subcellular compartment. J. Cell. Biochem. 108: 1252,1262, 2009. © 2009 Wiley-Liss, Inc. [source] Signaling mechanisms that regulate actin-based motility processes in the nervous systemJOURNAL OF NEUROCHEMISTRY, Issue 3 2002Gary Meyer Abstract Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development. The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood. [source] Dependence of axon initial segment formation on Na+ channel expressionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005Xiaorong Xu Abstract Spinal motor neurons were isolated from embryonic rats, and grown in culture. By 2 days in vitro, the axon initial segment was characterized by colocalization and clustering of Na+ channels and ankyrinG. By 5 days, NrCAM, and neurofascin could also be detected at most initial segments. We sought to determine, as one important aim, whether Na+ channels themselves played an essential role in establishing this specialized axonal region. Small hairpin RNAs (shRNAs) were used to target multiple subtypes of Na+ channels for reduced expression by RNA interference. Transfection resulted in substantial knockdown of these channels within the cell body and also as clusters at initial segments. Furthermore, Na+ currents originating at the initial segment, and recorded under patch clamp, were strongly reduced by shRNA. Control shRNA against a nonmammalian protein was without effect. Most interestingly, targeting Na+ channels also blocked clustering of ankyrinG, NrCAM, and neurofascin at the initial segment, although these proteins were seen in the soma. Thus, both Na+ channels and ankyrinG are required for formation of this essential axonal domain. Knockdown of Na+ channels was somewhat less effective when introduced after the initial segments had formed. Disruption of actin polymerization by cytochalasin D resulted in multiple initial segments, each with clusters of both Na+ channels and ankyrinG. The results indicate that initial segment formation occurs as Na+ channels are transported into the nascent axon membrane, diffuse distally, and link to the cytoskeleton by ankyrinG. Subsequently, other components are added, and stability is increased. A computational model closely reproduced the experimental results. © 2005 Wiley-Liss, Inc. [source] A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activationJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2005C. SOULET Summary., The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a Gq -dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by Fc,RIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the ,2A -adrenergic receptor/Gz pathway by epinephrine was able to replace the P2Y12/Gi -mediated pathway to amplify Rac activation by Fc,RIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein ,, subunits scavenger peptide. [source] CC Chemokine Receptor 4 (CCR4) in human allergen-induced late nasal responsesALLERGY, Issue 9 2010G. Banfield To cite this article: Banfield G, Watanabe H, Scadding G, Jacobson MR, Till SJ, Hall DA, Robinson DS, Lloyd CM, Nouri-Aria KT, Durham SR. CC Chemokine Receptor 4 (CCR4) in human allergen-induced late nasal responses. Allergy 2010; 65: 1126,1133. Abstract Background:, CC Chemokine receptor 4 (CCR4) is preferentially expressed on Th2 lymphocytes. CCR4-mediated inflammation may be important in the pathology of allergic rhinitis. Disruption of CCR4 , ligand interaction may abrogate allergen-induced inflammation. Methods:, Sixteen allergic rhinitics and six nonatopic individuals underwent both allergen and control (diluent) nasal challenges. Symptom scores and peak nasal inspiratory flow were recorded. Nasal biopsies were taken at 8 h post challenge. Sections were immunostained and examined by light or dual immunofluorescence microscopy for eosinophils, T-lymphocytes, CCR4+CD3+ and CXCR3+CD3+ cells and examined by in situ hybridization for CCR4, IL-4 and IFN-, mRNA+ cells. Peripheral blood mononuclear cells were obtained from peripheral blood of nine normal donors and the CCR4+CD4+ cells assessed for actin polymerization in response to the CCR4 ligand macrophage-derived chemokine (MDC/CCL22) and the influence of a CCR4 antagonist tested. Results:, Allergic rhinitics had increased early and late phase symptoms after allergen challenge compared to diluent; nonatopics did not respond to either challenge. Eosinophils, but not total numbers of CD3+ T cells, were increased in rhinitics following allergen challenge. In rhinitics, there was an increase in CCR4+CD3+ protein-positive cells relative to CXCR3+CD3+ cells; CCR4 mRNA+ cells were increased and IL-4 increased to a greater extent than IFN-,. CCR4+CD4+ T cells responded to MDC in vitro, and this response was inhibited by the selective CCR4 antagonist. Conclusion:, Lymphocyte CCR4 expression is closely associated with induction of human allergen-induced late nasal responses. Blocking CCR4-ligand interaction may provide a novel therapeutic approach in allergic disease. [source] Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25, T cells from atopic and nonatopic donorsALLERGY, Issue 8 2009D. Ahern Background:, Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown. Objective:, To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25, T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro. Methods:, Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25, T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling. Results:, CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25, effector cells, which downregulated CCR7 and upregulated CCR4. Conclusions:, CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25, effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells. [source] Soluble vascular cell adhesion molecule-1 induces human eosinophil migrationALLERGY, Issue 5 2009S. Ueki Background:, Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses including asthma. Adhesion molecules are known to play an important role in the accumulation of eosinophils in allergic inflammatory foci, and they contribute to eosinophil activation. Elevated levels of the soluble forms of adhesion molecules in the body fluid of asthmatic patients have been observed, although their pathophysiological significance remains to be fully elucidated. Methods:, Peripheral blood eosinophils were purified, and the effect of soluble vascular cell adhesion molecule-1 (sVCAM-1) on eosinophil migration was investigated using in vitro systems. Results:, We found that sVCAM-1 (1 to 10 ,g/ml) induced eosinophil chemotaxis, rather than chemokinesis, in a concentration-dependent fashion. In addition, sVCAM-1 induced cell shape change and actin polymerization, which are necessary for cell movement. Manipulations with very late antigen (VLA)-4-neutralizing antibody and signal inhibitors indicated that the sVCAM-1-induced chemotaxis was mediated through ligand-dependent activation of tyrosine kinase Src, p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK) MAPK. Rapid phosphorylation of these signaling molecules was observed using a bead-based multiplex assay. Conclusion:, Our results raise the possibility of sVCAM-1 in the fluid phase as a significant contributor to the heightened eosinophilic inflammatory response. [source] Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombeMOLECULAR MICROBIOLOGY, Issue 4 2008Ma Antonia Villar-Tajadura Summary Schizosaccharomyces pombe Rho2 GTPase regulates ,-D-glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase-activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor-stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N-terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this GTPase. Accordingly, rga2, cells contain more ,-D-glucan and therefore partially suppress the thermosensitivity of mok1,664 cells, which have a defective ,-D-glucan synthase. Additionally, genetic interactions and biochemical analysis suggest that Rga2 regulates Rho2,Pck2 interaction and might participate in the regulation of the MAPK cell integrity pathway. [source] Self-association of EPEC intimin mediated by the ,-barrel-containing anchor domain: a role in clustering of the Tir receptorMOLECULAR MICROBIOLOGY, Issue 1 2004Thierry Touzé Summary Outer membrane intimin directs attachment of enteropathogenic Escherichia coli (EPEC) via its Tir receptor in mammalian target cell membranes. Phosphorylation of Tir triggers local actin polymerization and the formation of ,pedestal-like' pseudopods. We demonstrate that the intimin protein contains three domains, a flexible N-terminus (residues 40,188), a central membrane-integrated ,-barrel (189,549), and a tightly folded Tir-binding domain (550,939). Intimin was shown by electron microscopy to form ring-like structures with a ,7 nm external diameter and an electron dense core, and to form channels of 50picoSiemens conductance in planar lipid bilayers. Gel filtration, multiangle light scattering and cross-linking showed that this central ,-barrel membrane-anchoring domain directs intimin dimerization. Isothermal titration calorimetry revealed a high affinity, single-binding site interaction of 2 : 1 stoichiometry between dimeric intimin and Tir, and modelling suggests that this interaction determines a reticular array-like superstructure underlying receptor clustering. In support of this model, actin rearrangement induced in Tir-primed cultured cells by intimin-containing proteoliposomes was dependent on the concentration of both intimin and Tir, and co-localized with clustered phosphorylated Tir. [source] Altered actin polymerization of Plutella xylostella (L.) in response to ovarian calyx components of an endoparasitoid Cotesia plutellae (Kurdjumov)PHYSIOLOGICAL ENTOMOLOGY, Issue 2 2009MADANAGOPAL NALINI AbstractCotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte-spreading behaviour of P. xylostella is analysed by measuring F-actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte-spreading peptide, in a dose-dependent manner for each calyx component and fail to exhibit haemocyte-spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte-spreading could be explained by measuring F-actin contents, in which parasitization by C. plutellae inhibits F-actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F-actin development in the nonparasitized haemocytes. In addition, co-incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte-spreading and F-actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization. [source] On-line confocal imaging of the events leading to structural dedifferentiation of an axonal segment into a growth cone after axotomyTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2006Iman Sahly Abstract The transformation of a transected axonal tip into a growth cone (GC) after axotomy is a critical step in the cascade of events leading to regeneration. However, the mechanisms underlying it are largely unknown. In earlier studies we reported that axotomy of cultured Aplysia neurons leads to a transient and local increase in the free intracellular Ca2+ concentration, calpain activation, and localized proteolysis of the submembranal spectrin. In a recent ultrastructural study, we reported that calpain activation is critical for the restructuring of the microtubules and neurofilaments at the cut axonal end to form a compartment in which vesicles accumulate. By using on-line confocal imaging of microtubules (MTs), actin, and vesicles in cultured Aplysia neurons, we studied the kinetics of the transformation and examined some of the mechanisms that orchestrate it. We report that perturbation of the MTs' polymerization by nocodazole inhibits the formation of an MT-based compartment in which the vesicles accumulate, yet actin repolymerization proceeds normally to form a nascent GC's lamellipodium. Nevertheless, under these conditions, the lamellipodium fails to expand and form neurites. When actin filament polymerization is inhibited by cytochalasin D or jasplakinolide, the MT-based compartment is formed and vesicles accumulate at the cut axonal end. However, a GC's lamellipodium is not formed, and the cut axonal end fails to regenerate. A growth-competent GC is formed only when MT restructuring, the accumulation of vesicles, and actin polymerization properly converge in time and space. J. Comp. Neurol. 494:705,720, 2006. © 2005 Wiley-Liss, Inc. [source] Dynamic association between ,-actinin and ,-integrin regulates contraction of canine tracheal smooth muscleTHE JOURNAL OF PHYSIOLOGY, Issue 3 2006Wenwu Zhang The adhesion junctions of smooth muscle cells may be dynamically regulated during smooth muscle contraction, and this dynamic regulation may be important for the development of active tension. In the present study, the role of ,-actinin during smooth muscle contraction was evaluated in tracheal smooth muscle tissues and freshly dissociated cells. Stimulation with acetylcholine (ACh) increased the localization of ,-actinin at the membrane of freshly dissociated smooth muscle cells, and increased the amount of ,1 integrin that coprecipitated with ,-actinin from muscle tissue homogenates. GFP-,-actinin fusion proteins were expressed in muscle tissues and visualized in live freshly dissociated cells. GFP-,-actinin translocated to the membrane within seconds of stimulation of the cells with ACh. Expression of the integrin-binding rod domain of ,-actinin in smooth muscle tissues depressed active contraction in response to ACh. Expression of the ,-actinin rod domain also inhibited the translocation of endogenous ,-actinin to the membrane, and inhibited the association of endogenous ,-actinin with ,1 -integrin in ,-actinin immunoprecipitates from tissue extracts. However, the expression of ,-actinin rod domain peptides did not inhibit increases in myosin light chain phosphorylation or actin polymerization in response to stimulation with ACh. Results suggest that contractile stimulation of smooth muscle causes the rapid recruitment of ,-actinin to ,-integrin complexes at the membrane, and that the recruitment of ,-actinin to integrin complexes is necessary for active tension development in smooth muscle. [source] Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin BTHE PLANT JOURNAL, Issue 2 2006Yanmei Chen Summary To investigate roles of the actin cytoskeleton in growth of the pollen tube of Picea meyeri, we used the actin polymerization inhibitor latrunculin B (LATB) under quantitatively controlled conditions. At low concentrations, LATB inhibited polymerization of the actin cytoskeleton in the growing pollen tube, which rapidly inhibited tip growth. The proteomic approach was used to analyse protein expression-profile changes during pollen germination and subsequent pollen-tube development with disturbed organization of the actin cytoskeleton. Two-dimensional electrophoresis and staining with Coomassie Brilliant Blue revealed nearly 600 protein spots. A total of 84 of these were differentially displayed at different hours with varying doses of LATB, and 53 upregulated or downregulated proteins were identified by mass spectrometry. These proteins were grouped into distinct functional categories including signalling, actin cytoskeleton organization, cell expansion and carbohydrate metabolism. Moreover, actin disruption affected the morphology of Golgi stacks, mitochondria and amyloplasts, along with a differential expression of proteins involved in their functions. These findings provide new insights into the multifaceted mechanism of actin cytoskeleton functions and its interaction with signalling, cell-expansion machinery and energy-providing pathways. [source] The polycomb group protein EZH2 regulates actin polymerization in human prostate cancer cellsTHE PROSTATE, Issue 3 2008R.J. Bryant Abstract BACKGROUND The Polycomb Group protein EZH2 is implicated in prostate cancer progression. EZH2 promotes prostate cancer cell proliferation and invasiveness. We describe a link between EZH2 function and actin polymerization in prostate cancer cells. METHODS Nuclear and cytoplasmic EZH2 expression in benign and malignant prostate tissue samples was assessed. An association between EZH2 function and actin polymerization in prostate cancer cells was investigated using siRNA-mediated knock-down of EZH2. Effects of EZH2 knock-down on actin polymerization dynamics were analyzed biochemically using immunoblot analysis of cell lysate fractions, and morphologically using immunocytochemistry. RESULTS Cytoplasmic EZH2 is expressed at low levels in benign prostate epithelial cells and over-expressed in prostate cancer cells. Cytoplasmic EZH2 expression levels correlate with nuclear EZH2 expression in prostate cancer samples. Knock-down of EZH2 in PC3 prostate cancer cells increases the amount of F-actin polymerization, cell size, and formation of actin-rich filaments. CONCLUSIONS Cytoplasmic EZH2 is over-expressed in prostate cancer cells. EZH2 function promotes a reduction in the pool of insoluble F-actin in invasive prostate cancer cells. EZH2 may regulate actin polymerization dynamics and thereby promote prostate cancer cell motility and invasiveness. Prostate 68: 255,263, 2008. © 2007 Wiley-Liss, Inc. [source] Angiopoietin 1 directly induces destruction of the rheumatoid joint by cooperative, but independent, signaling via ERK/MAPK and phosphatidylinositol 3-kinase/AktARTHRITIS & RHEUMATISM, Issue 7 2007Akira Hashiramoto Objective To determine whether angiopoietin 1 (Ang-1) potentiates overgrowth of the synovium and joint degradation in rheumatoid arthritis (RA), and to clarify the cell-signaling mechanisms of Ang-1 in the rheumatoid joint. Methods Expression of Ang-1, TIE-2 (a receptor for Ang-1), and matrix metalloproteinase 3 (MMP-3) was studied by immunohistochemistry. Activation of the ERK/MAPK and phosphatidylinositol (PI) 3-kinase/Akt pathways and of NF-,B was determined by Western blotting and an NF-,B p65 DNA binding activity assay, respectively. Induction of apoptosis was evaluated by nuclear staining, cell viability assay, and Western blotting of caspases. Synovial cell migration was evaluated by actin polymerization, Western blotting of Rho family proteins, and affinity purification with Rhotekin-Rho and p21-activated kinase 1. Matrix degradation was examined by induction of proMMP-3 secretion from synovial cells followed by in vitro cartilaginous matrix degradation assay. Results Ang-1 stimulated the ERK/MAPK and PI 3-kinase/Akt pathways in a cooperative but independent manner, which enhanced rheumatoid synovium overgrowth and joint destruction. In addition, Ang-1 activated NF-,B via Akt to promote cell growth, but also inhibited cell apoptosis via ERK and Akt. Ang-1 directly potentiated the extension of synovial cells in an ERK- and Akt-dependent manner by up-regulating Rho family proteins, which attenuated Rac signaling and led to membrane ruffling. Ang-1 induced proMMP-3 secretion from synovial cells, which resulted in direct degradation of the cartilaginous matrix. Conclusion Ang-1 stimulates the ERK/MAPK and PI 3-kinase/Akt pathways cooperatively, but in a manner independent of each other, to directly potentiate synovium overgrowth and joint destruction in RA. In addition to inflammatory cytokines, Ang-1/TIE-2 signaling appears to be an independent factor that contributes to the destruction of the rheumatoid joint. [source] Functional importance of the actin cytoskeleton in contraction of bovine iris sphincter muscleAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 3 2002J. A. C. Filipe Summary 1 The contractile capacity of smooth muscle cells depends on the cytoskeletal framework of the cell. The aim of this study was to determine the functional importance of both the actin and the tubulin components of the cytoskeleton in contractile responses of the bovine isolated iris sphincter muscle. 2 In each preparation, two contractions to the muscarinic agonist carbachol were obtained. The maximum responses of the first contractions were taken as 100%. The second contractions to carbachol were elicited in the presence of either cytochalasin B (50 and 5 ,m), an inhibitor of the actin cytoskeleton, or colchicine (100 ,m), an inhibitor of the tubulin cytoskeleton (30 min incubation). 3 Cytochalasin B, at a concentration of 50 ,m, significantly decreased the contractions induced by carbachol, with the maximum response reduced to 21.8 ± 6.6% (n = 12) of the initial maximum. The maximal contractions to carbachol in the presence of colchicine reached 96.2 ± 7.9% (n = 9) of the initial contraction, which was not significantly different from control second responses to carbachol with neither drug present, which reached 113.3 ± 7.6% (n = 7). 4 The effect of cytochalasin B was dose-dependent, since at a lower concentration of 5 ,m, the drug decreased the maximum contraction to carbachol to 60.3 ± 8.8% (n = 6). The effect of cytochalasin B was at least partially reversible, since after the use of the higher concentration of 50 ,m, contractions to carbachol increased to 62.3 ± 15.5% (n = 4) of the maximal response, after 1 h repeated washing of the preparations. 5 Cytochalasin D, at a concentration of 50 ,m, completely abolished the contractions induced by carbachol (n = 4). 6 These findings suggest that in bovine iris sphincter muscle, contractions to carbachol are highly dependent, from a functional point of view, on actin polymerization, and not, to any important degree, on the polymerization of tubulin. [source] Use of Green Fluorescent Protein-Conjugated ,-Actin as a Novel Molecular Marker for in Vitro Tumor Cell Chemotaxis AssayBIOTECHNOLOGY PROGRESS, Issue 6 2000Louis Hodgson To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector expressing a ,-actin-GFP fusion protein was generated. The expression construct when transfected into NIH3T3 fibroblast, A2058 human melanoma and 293T human embryonic kidney carcinoma cell lines expressed ,-actin-GFP fusion protein, which colocalized with endogenous cellular actin as determined by histoimmunofluorescence staining. The ,-actin-GFP was also observed to be reorganized in response to treatments with the chemoattractant type IV collagen. Cells extended pseudopodial protrusions and altered the morphology of their cortical structure in response to type IV collagen stimulation. More importantly, ,-actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that ,-actin-GFP could participate in actin polymerization. Although ectopic expression of ,-actin-GFP lead to minor side effects on cell proliferation, these studies suggest that this strategy provides an alternative to the invasive techniques currently used to study actin dynamics and permits real-time visualization of actin rearrangements in response to environmental cues. [source] | |||||||||||||||||||||||||