scholarly journals Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells

2001 ◽  
Vol 155 (3) ◽  
pp. 331-338 ◽  
Author(s):  
Martin Villalba ◽  
Kun Bi ◽  
Fernando Rodriguez ◽  
Yoshihiko Tanaka ◽  
Stephen Schoenberger ◽  
...  
Keyword(s):  
T Cells ◽  
Actin Cytoskeleton ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Large Scale ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Membrane Microdomains ◽  
Jurkat T Cells ◽  
Cytoskeleton Reorganization

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1−/−) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.

Signaling through ZAP-70 is required for CXCL12-mediated T-cell transendothelial migration

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3111-3118 ◽  
Author(s):  
Michel Ticchioni ◽  
Céline Charvet ◽  
Nelly Noraz ◽  
Laurence Lamy ◽  
Marcos Steinberg ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Cross Talk ◽  
Actin Polymerization ◽  
Transendothelial Migration ◽  
Dominant Negative ◽  
Jurkat T Cells ◽  
T Cell Migration ◽  
Pharmacologic Inhibition

Abstract Transendothelial migration of activated lymphocytes from the blood into the tissues is an essential step for immune functions. The housekeeping chemokine CXCL12 (or stroma cell–derived factor-1α), a highly efficient chemoattractant for T lymphocytes, drives lymphocytes to sites where they are highly likely to encounter antigens. This suggests that cross-talk between the T-cell receptor (TCR) and CXCR4 (the CXCL12 receptor) might occur within these sites. Here we show that the zeta-associated protein 70 (ZAP-70), a key element in TCR signaling, is required for CXCR4 signal transduction. The pharmacologic inhibition of ZAP-70, or the absence of ZAP-70 in Jurkat T cells and in primary CD4+ T cells obtained from a patient with ZAP deficiency, resulted in an impairment of transendothelial migration that was rescued by the transfection of ZAP-70. Moreover, the overexpression of mutated forms of ZAP-70, whose kinase domain was inactivated, also abrogated the migratory response of Jurkat T cells to CXCL12. In contrast, no involvement of ZAP-70 in T-cell arrest on inflammatory endothelium under flow conditions or in CXCL12-induced actin polymerization was observed. Furthermore, CXCL12 induced time-dependent phosphorylation of ZAP-70, Vav1, and extracellular signal-regulated kinases (ERKs); the latter were reduced in the absence of functional ZAP-70. However, though a dominant-negative Vav1 mutant (Vav1 L213A) blocked CXCL12-induced T-cell migration, pharmacologic inhibition of the ERK pathway did not affect migration, suggesting that ERK activation is dispensable for T-cell chemotaxis. We conclude that cross-talk between the ZAP-70 signaling pathway and the chemokine receptor CXCR4 is required for T-cell migration.

scholarly journals Rab5 Induces Rac-independent Lamellipodia Formation and Cell Migration

1999 ◽  
Vol 10 (10) ◽  
pp. 3239-3250 ◽  
Author(s):  
Marcel Spaargaren ◽  
Johannes L. Bos
Keyword(s):  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Strong Support ◽  
Leading Edge ◽  
Vesicle Fusion ◽  
Dominant Negative ◽  
Cytoskeleton Reorganization ◽  
Endocytic Vesicles ◽  
Lamellipodia Formation

Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative Rab5 inhibits vesicle fusion. Here we show that introduction of active Rab5 in fibroblasts also induced reorganization of the actin cytoskeleton but not of microtubule filaments, resulting in prominent lamellipodia formation. The Rab5-induced lamellipodia formation did not require activation of PI3-K or the GTPases Ras, Rac, Cdc42, or Rho, which are all strongly implicated in cytoskeletal reorganization. Furthermore, lamellipodia formation by insulin, Ras, or Rac was not affected by expression of dominant negative Rab5. In addition, cells expressing active Rab5 displayed a dramatic stimulation of cell migration, with the lamellipodia serving as the leading edge. Both lamellipodia formation and cell migration were dependent on actin polymerization but not on microtubules. These results demonstrate that Rab5 induces lamellipodia formation and cell migration and that the Rab5-induced lamellipodia formation occurs by a novel mechanism independent of, and distinct from, PI3-K, Ras, or Rho-family GTPases. Thus, Rab5 can control not only endocytosis but also actin cytoskeleton reorganization and cell migration, which provides strong support for an intricate relationship between these processes.

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.

Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation

2002 ◽  
Vol 115 (12) ◽  
pp. 2603-2611 ◽  
Author(s):  
Martha Triantafilou ◽  
Kensuke Miyake ◽  
Douglas T. Golenbock ◽  
Kathy Triantafilou
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Cell Activation ◽  
Imaging Techniques ◽  
Recognition System ◽  
Innate Immune ◽  
Membrane Microdomains ◽  
Immune Recognition ◽  
Cellular Activation ◽  
Signalling Molecules

The plasma membrane of cells is composed of lateral heterogeneities,patches and microdomains. These membrane microdomains or lipid rafts are enriched in glycosphingolipids and cholesterol and have been implicated in cellular processes such as membrane sorting and signal transduction. In this study we investigated the importance of lipid raft formation in the innate immune recognition of bacteria using biochemical and fluorescence imaging techniques. We found that receptor molecules that are implicated in lipopolysaccharide (LPS)-cellular activation, such as CD14, heat shock protein(hsp) 70, 90, Chemokine receptor 4 (CXCR4), growth differentiation factor 5(GDF5) and Toll-like receptor 4 (TLR4), are present in microdomains following LPS stimulation. Lipid raft integrity is essential for LPS-cellular activation, since raft-disrupting drugs, such as nystatin or MCD, inhibit LPS-induced TNF-α secretion. Our results suggest that the entire bacterial recognition system is based around the ligation of CD14 by bacterial components and the recruitment of multiple signalling molecules, such as hsp70, hsp90, CXCR4, GDF5 and TLR4, at the site of CD14-LPS ligation, within the lipid rafts.

scholarly journals Palmitoylation of the Autographa californica Multicapsid Nucleopolyhedrovirus Envelope Glycoprotein GP64: Mapping, Functional Studies, and Lipid Rafts

2003 ◽  
Vol 77 (11) ◽  
pp. 6265-6273 ◽  
Author(s):  
Sandy Xiaoxin Zhang ◽  
Yu Han ◽  
Gary W. Blissard
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Envelope Glycoprotein ◽  
Cysteine Residue ◽  
Autographa Californica ◽  
Membrane Microdomains ◽  
Sf9 Cells ◽  
Wild Type ◽  
Lipid Raft Microdomains

ABSTRACT Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.

scholarly journals Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes

1998 ◽  
Vol 66 (11) ◽  
pp. 5527-5533 ◽  
Author(s):  
Sonia Meconi ◽  
Véronique Jacomo ◽  
Patrice Boquet ◽  
Didier Raoult ◽  
Jean-Louis Mege ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Coxiella Burnetii ◽  
Actin Polymerization ◽  
Q Fever ◽  
Morphological Changes ◽  
Critical Role ◽  
Filamentous Actin ◽  
Cytoskeleton Reorganization ◽  
Membrane Protrusions ◽  
Actin Protrusions

ABSTRACT Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetiiorganisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetiimay therefore play a critical role in the internalization strategy of this bacterium.

Lipid raft adhesion receptors and Syk regulate selectin-dependent rolling under flow conditions

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3352-3359 ◽  
Author(s):  
Claire Abbal ◽  
Martine Lambelet ◽  
Debora Bertaggia ◽  
Carole Gerbex ◽  
Manuel Martinez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Chelating Agents ◽  
Regulatory Mechanisms ◽  
Membrane Microdomains ◽  
Leukocyte Rolling ◽  
Flow Conditions ◽  
Cell Rolling ◽  
Pharmacologic Inhibitors

Abstract Selectins and their ligand P-selectin glycoprotein ligand-1 (PSGL-1) mediate leukocyte rolling along inflamed vessels. Cell rolling is modulated by selectin interactions with their ligands and by topographic requirements including L-selectin and PSGL-1 clustering on tips of leukocyte microvilli. Lipid rafts are cell membrane microdomains reported to function as signaling platforms. Here, we show that disruption of leukocyte lipid rafts with cholesterol chelating agents depleted raft-associated PSGL-1 and L-selectin and strongly reduced L-, P-, and E-selectin–dependent rolling. Cholesterol repletion reversed inhibition of cell rolling. Importantly, leukocyte rolling on P-selectin induced the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase associated to lipid raft PSGL-1. Furthermore, inhibition of Syk activity or expression, with pharmacologic inhibitors or by RNA interference, strongly reduced leukocyte rolling on P-selectin, but not on E-selectin or PSGL-1. These observations identify novel regulatory mechanisms of leukocyte rolling on selectins with a strong dependency on lipid raft integrity and Syk activity.

scholarly journals Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds

2013 ◽  
Vol 16 (6) ◽  
pp. 1361-1371 ◽  
Author(s):  
Caroline Nothdurfter ◽  
Sascha Tanasic ◽  
Barbara Di Benedetto ◽  
Manfred Uhr ◽  
Eva-Maria Wagner ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Lipid Rafts ◽  
Gabaa Receptor ◽  
Lipid Raft ◽  
Tricyclic Antidepressant ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Receptor Modulation ◽  
Gabaa Receptor Subunits ◽  
Triton X

Abstract Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

Lysophosphatidic acid-mediated augmentation of cardiomyocyte lipoprotein lipase involves actin cytoskeleton reorganization

2005 ◽  
Vol 288 (6) ◽  
pp. H2802-H2810 ◽  
Author(s):  
Thomas Pulinilkunnil ◽  
Ding An ◽  
Sanjoy Ghosh ◽  
Dake Qi ◽  
Girish Kewalramani ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Lipoprotein Lipase ◽  
Lysophosphatidic Acid ◽  
Actin Polymerization ◽  
Contractile Function ◽  
Rho Kinase ◽  
Filamentous Actin ◽  
Membrane Translocation ◽  
Downstream Effector ◽  
Cytoskeleton Reorganization

The lipoprotein lipase (LPL)-augmenting property of lysophosphatidylcholine requires the formation of lysophosphatidic acid (LPA) ( J Mol Cell Cardiol 37: 931–938, 2004). Given that the actin cytoskeleton has been implicated in regulating cardiomyocyte LPL, we examined whether LPL secretion after LPA involves actin cytoskeleton reassembly. Incubation of myocytes with LPA (1–100 nM) increased basal and heparin-releasable LPL (HR-LPL), an effect that was independent of shifts in LPL mRNA. The influence of LPA on myocyte LPL was reflected at the coronary lumen, with substantial increases of the enzyme at this location. Incubation of myocytes with cytochalasin D not only blocked LPA-induced augmentation of HR-LPL but also abrogated filamentous actin formation. These effects of LPA were likely receptor mediated. Exposure of myocytes to LPA facilitated significant membrane translocation of RhoA and its downstream effector Rho kinase I (ROCK I), and blocking this effect with Y-27632 appreciably reduced basal and HR-LPL activity. Incubation of adipose tissue with LPA also significantly enhanced basal and HR-LPL activity, suggesting that sarcomeric actin likely has a limited role in influencing the LPL secretory function of LPA in the myocyte. Comparable to LPA, hyperglycemia also caused significant membrane translocation of RhoA and ROCK I in hearts isolated from diazoxide-treated animals, effects that were abrogated using insulin. Overall, our data suggest that comparable to hyperglycemia, LPA-induced increases in cardiac LPL occurred via posttranscriptional mechanisms and processes that likely required RhoA activation and actin polymerization. Whether this increase in LPL augments triglyceride deposition in the heart leading to eventual impairment in contractile function is currently unknown.

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