scholarly journals Microvilli-like structures are associated with the internalization of virulent capsulatedNeisseria meningitidisinto vascular endothelial cells

2002 ◽  
Vol 115 (6) ◽  
pp. 1231-1241 ◽  
Author(s):  
Emmanuel Eugène ◽  
Isabelle Hoffmann ◽  
Céline Pujol ◽  
Pierre-Olivier Couraud ◽  
Sandrine Bourdoulous ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Vascular Endothelial Cells ◽  
Transmembrane Proteins ◽  
Bacterial Attachment ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Phagocytic Cells ◽  
Type Iv

Bacterial pathogens are internalized into non-phagocytic cells either by a zipper mechanism involving a direct contact between a bacterial ligand and a cellular receptor or a trigger mechanism secondary to the formation of membrane ruffles. Here we show that internalization of capsulated Neisseria meningitidis within endothelial cells following type IV pilus-mediated adhesion is associated with the formation of cellular protrusions at the site of bacterial attachment. These protrusions, like microvilli, are highly enriched in ezrin and moesin, two members of the ERM(ezrin/radixin/moesin) family, whereas vinculin and paxillin are absent. ERM-binding transmembrane proteins, such as CD44, and cortical actin polymerization colocalized within these membrane protrusions. Expression of dominant-negative ezrin largely prevented cortical actin polymerization, thus confirming the role of this molecule in bacteria-induced cytoskeletal modifications. Moreover, using selective inhibitors and dominant-negative mutants of the Rho family GTPases, we show that bacteria-induced actin polymerization required the activation of both Rho and Cdc42 but not of Rac1. Whereas GTPase inhibition dramatically reduced actin polymerization at the site of bacterial attachment, ezrin recruitment was not affected, indicating that bacterial adhesion promotes ezrin recruitment independently of the activity of the Rho-GTPases. Furthermore, GTPase inhibition largely reduced N. meningitidis entry into endothelial cells without affecting adhesion. We thus propose that following pilus-mediated adhesion, capsulated N. meningitidis recruit ERM-binding transmembrane proteins, as well as ezrin and moesin, and that both Rho and Cdc42 are critical for the subsequent cytoskeletal modifications responsible for the formation of microvilli-like cellular protrusions and bacterial internalization.

scholarly journals Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

2011 ◽  
Vol 208 (5) ◽  
pp. 1055-1068 ◽  
Author(s):  
Bebhinn Treanor ◽  
David Depoil ◽  
Andreas Bruckbauer ◽  
Facundo D. Batista
Keyword(s):  
Actin Cytoskeleton ◽  
B Cell ◽  
Protein Function ◽  
Actin Polymerization ◽  
Lymphocyte Activation ◽  
Dominant Negative ◽  
Temporary Increase ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Diffusion Dynamics

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells

2002 ◽  
Vol 115 (12) ◽  
pp. 2475-2484 ◽  
Author(s):  
Valérie Vouret-Craviari ◽  
Christine Bourcier ◽  
Etienne Boulter ◽  
Ellen Van Obberghen-Schilling
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Cell Spreading ◽  
Actin Binding ◽  
Sphingosine 1 Phosphate ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

scholarly journals RacG Regulates Morphology, Phagocytosis, and Chemotaxis

2006 ◽  
Vol 5 (10) ◽  
pp. 1648-1663 ◽  
Author(s):  
Baggavalli P. Somesh ◽  
Georgia Vlahou ◽  
Miho Iijima ◽  
Robert H. Insall ◽  
Peter Devreotes ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Free System ◽  
Mild Phenotype ◽  
Particle Uptake ◽  
Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

scholarly journals Podocalyxin is required for maintaining blood–brain barrier function during acute inflammation

2019 ◽  
Vol 116 (10) ◽  
pp. 4518-4527 ◽  
Author(s):  
Jessica Cait ◽  
Michael R. Hughes ◽  
Matthew R. Zeglinski ◽  
Allen W. Chan ◽  
Sabrina Osterhof ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Barrier Function ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Focal Adhesions ◽  
Brain Barrier ◽  
Cortical Actin ◽  
Inflammatory Conditions ◽  
Blood Brain

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele ofPodxlin vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood–brain barrier (BBB) inPodxlΔTie2Cremice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primedPodxlΔTie2Cremice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

scholarly journals The TRE17 Oncogene Encodes a Component of a Novel Effector Pathway for Rho GTPases Cdc42 and Rac1 and Stimulates Actin Remodeling

2003 ◽  
Vol 23 (6) ◽  
pp. 2151-2161 ◽  
Author(s):  
Jeffrey M. Masuda-Robens ◽  
Sara N. Kutney ◽  
Hongwei Qi ◽  
Margaret M. Chou
Keyword(s):  
Plasma Membrane ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Actin Remodeling ◽  
Membrane Recruitment ◽  
Truncation Mutant ◽  
Effector Pathway

ABSTRACT The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.

scholarly journals The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

1998 ◽  
Vol 143 (4) ◽  
pp. 1087-1099 ◽  
Author(s):  
Angeliki Malliri ◽  
Marc Symons ◽  
Robert F. Hennigan ◽  
Adam F.L. Hurlstone ◽  
Richard F. Lamb ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cortical Actin ◽  
A431 Cells ◽  
Membrane Ruffling ◽  
In Vitro Invasion ◽  
Cytoskeletal Rearrangements ◽  
Lamellipodia Formation

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Cross-Talk between Rho GTPases Regulates Actin Cytoskeleton and Chemotaxis of Hematopoietic Progenitor Cell.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 266-266
Author(s):  
Hee-Don Chae ◽  
Katherine E. Lee ◽  
Aparna C. Jasti ◽  
David A. Williams ◽  
Yi Gu
Keyword(s):  
Cell Membrane ◽  
Actin Cytoskeleton ◽  
Cross Talk ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Actin Assembly ◽  
Hematopoietic Progenitor ◽  
Stimulation Of

Abstract Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins. RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2]. To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.

scholarly journals IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways

2001 ◽  
Vol 280 (6) ◽  
pp. L1094-L1103 ◽  
Author(s):  
Ingrid U. Schraufstatter ◽  
Janice Chung ◽  
Meike Burger
Keyword(s):  
Endothelial Cells ◽  
Pertussis Toxin ◽  
Early Phase ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Microvascular Endothelial Cells ◽  
Cortical Actin ◽  
Microvascular Endothelial ◽  
Cell Retraction ◽  
Later Phase

Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to Gi. In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.

scholarly journals Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis

2001 ◽  
Vol 155 (1) ◽  
pp. 133-144 ◽  
Author(s):  
Isabelle Hoffmann ◽  
Emmanuel Eugène ◽  
Xavier Nassif ◽  
Pierre-Olivier Couraud ◽  
Sandrine Bourdoulous
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Actin Cytoskeleton ◽  
Neisseria Meningitidis ◽  
Receptor Tyrosine Kinase ◽  
Cellular Localization ◽  
Actin Polymerization ◽  
Egf Receptors ◽  
Type Iv ◽  
The Family

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus–mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.

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