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Verena Niggli - One of the best experts on this subject based on the ideXlab platform.
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analysis of close associations of Uropod associated proteins in human t cells using the proximity ligation assay
PeerJ, 2013Co-Authors: Tommy Baumann, Sarah Affentranger, Verena NiggliAbstract:We have shown previously that the raft-associated proteins flotillin-1 and -2 are rapidly recruited to the Uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Other proteins such as the adhesion receptor PSGL-1, the actin-membrane linker proteins ezrin/radixin/moesin (ERM) and the signaling enzyme phosphatidylinositol-4-phosphate 5-kinase type Iγ90 (PIPKIγ90) also accumulate in the T-cell Uropod. Using the in situ proximity ligation assay (PLA) we now have investigated putative close associations of these proteins in human freshly isolated T-cells before and after chemokine addition. The PLA allows in situ subcellular localization of close proximity of endogenous proteins at single-molecule resolution in fixed cells. It allows detection also of weaker and transient complexes that would not be revealed with co-immunoprecipitation approaches. We previously provided evidence for heterodimer formation of tagged flotillin-1 and -2 in T-cells before and after chemokine addition using fluorescence resonance energy transfer (FRET). We now confirm these findings using PLA for the endogenous flotillins in fixed human T-cells. Moreover, in agreement with the literature, our PLA findings confirm a close association of endogenous PSGL-1 and ERM proteins both in resting and chemokine-activated human T-cells. In addition, we provide novel evidence using the PLA for close associations of endogenous activated ERM proteins with PIPKIγ90 and of endogenous flotillins with PSGL-1 in human T-cells, before and after chemokine addition. Our findings suggest that preformed clusters of these proteins coalesce in the Uropod upon cell stimulation.
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determinants of phosphatidylinositol 4 phosphate 5 kinase type iγ90 Uropod location in t lymphocytes and its role in Uropod formation
PeerJ, 2013Co-Authors: Lucia Mathis, Sarah Affentranger, Sarah A Wernimont, Anna Huttenlocher, Verena NiggliAbstract:We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell Uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 Uropod targeting and the role that PIPKIγ90 plays in T cell Uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the Uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the Uropod. We further use T cells from PIPKIγ90(-/-) mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell Uropod formation. We find that PIPKIγ90 deficient T cells have elongated Uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses Uropod formation and impairs capping of Uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell Uropod formation.
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ezrin radixin moesin proteins and flotillins cooperate to promote Uropod formation in t cells
Frontiers in Immunology, 2013Co-Authors: Sibylla Martinelli, Janis K. Burkhardt, Emily J H Chen, Fiona Clarke, Ruth Lyck, Sarah Affentranger, Verena NiggliAbstract:T cell Uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell Uropod. Expression of wild-type ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced Uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and Uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant negative deletion mutant of ezrin, 1-310 ezrin-EGFP, markedly reduced Uropod formation induced by the chemokine SDF-1, T cell front-tail polarity and capping of PSGL-1 and flotillins. Transfection of T cells with wild-type or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1-310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and Uropod formation, indicating that ERM proteins and flotillins cooperate in Uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the Uropod of migrating T cells.
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flotillins and erm proteins function to promote Uropod formation in t cells 44 10
Journal of Immunology, 2010Co-Authors: Janis K. Burkhardt, Verena Niggli, Emily J H Chen, Meredith H ShafferAbstract:Migrating and adherent T cells form a protruding leading edge and a constricted tail-like structure termed the Uropod. The formation of these two structures involves the segregation of specific cytoskeletal elements and cell surface molecules, including proteins that organize cell polarity in other systems. Among the proteins that are segregated to the Uropod are ezrin and moesin, ERM family proteins that organize cell membrane domains by linking cytoplasmic and cytosolic proteins to the actin cytoskeleton. Using conditional ezrin-deficient mice in conjunction with siRNA for moesin, we show that ezrin and moesin are required for Uropod formation. In addition to ERM proteins, we find that the lipid-raft associated actin-binding proteins flotillins/reggies are also associated with the T cell Uropod. Flotillins form a polarized cap even under conditions where a constricted Uropod has not formed. Suppression of flotillin expression inhibits Uropod formation and the polarization of ERM proteins and their binding partners. These data support a model in which flotillins mark a cell surface domain to which ERM proteins are recruited, and ERM proteins then promote formation of a constricted Uropod structure. Since flotillins are not known to interact directly with ERM proteins, intermediary molecules, such as their common binding partner, PSGL-1, may be involved.
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flotillins interact with psgl 1 in neutrophils and upon stimulation rapidly organize into membrane domains subsequently accumulating in the Uropod
PLOS ONE, 2009Co-Authors: Jeremie Rossy, Dominique Schlicht, Britta Engelhardt, Verena NiggliAbstract:BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the Uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the Uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as Uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other Uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil Uropod formation and/or stabilization and organize Uropod localization of PSGL-1.
Masakazu Takahata - One of the best experts on this subject based on the ideXlab platform.
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neuronal mechanisms underlying the facilitatory control of Uropod steering behaviour during treadmill walking in crayfish i antagonistically regulated background excitability of Uropod motoneurones
The Journal of Experimental Biology, 1998Co-Authors: Maki Murayama, Masakazu TakahataAbstract:One of the postural reflexes of crayfish, the Uropod steering response, is elicited by specific sensory inputs while the animal is walking. It is not elicited, however, by the same inputs when the animal is at rest. To clarify the neuronal mechanisms underlying this facilitatory control of body posture in the active animals, we used intracellular recordings to analyse the synaptic activities of Uropod motor system neurones in an unanaesthetized whole-animal preparation. Several Uropod motoneurones were found to receive sustained depolarizing inputs during walking, whereas the walking leg motoneurones sampled always showed rhythmic activity. The membrane conductance of the Uropod motoneurones increased during the sustained synaptic activity. Premotor nonspiking interneurones showed depolarizing or hyperpolarizing membrane potential changes during walking that were also accompanied by increases in membrane conductance. Some of these interneurones enhanced Uropod motoneurone activity, whereas others suppressed it during walking. These results suggest that the background excitability of Uropod motoneurones is kept at an intermediate level during walking by the antagonistic inputs from premotor nonspiking interneurones so that the Uropod motor system can be responsive to both further excitatory and inhibitory inputs resulting from postural changes.
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neuronal mechanisms underlying the facilitatory control of Uropod steering behaviour during treadmill walking in crayfish ii modulation of Uropod motoneurone excitation by leg proprioception
The Journal of Experimental Biology, 1998Co-Authors: Maki Murayama, Masakazu TakahataAbstract:The synaptic activities underlying the Uropod steering behaviour of crayfish evoked by tilting the substratum beneath the legs have been studied intracellularly in unanaesthetized animals standing or walking on a treadmill. The Uropod motoneurones showed little or no synaptic response when the treadmill was tilted while the animal was in a quiescent state and the membrane potential was at its resting value. When the same stimulus was given while the animal was walking or in an active stance on the treadmill, the motoneurones showed transient much-enhanced excitatory or inhibitory responses to tilt, depending on the tilt direction. These responses were superimposed on a sustained level of background excitation so that the spike activity of the motoneurones either increased or decreased. Premotor nonspiking interneurones also showed little or no synaptic response to the tilt stimulus while the animal was resting, but greatly enhanced responses, in either a depolarizing or a hyperpolarizing direction, while the animal was walking or in the active-standing state. The results indicate that the proprioceptor inputs converging onto the Uropod motoneurones, either directly or through premotor nonspiking interneurones, are gated not only in the Uropod motor system in the terminal abdominal ganglion but also at as yet unidentified sites upstream in anterior ganglia, thus suggesting multiple gate control of the descending proprioceptor pathway.
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cholinergic transmission at mechanosensory afferents in the crayfish terminal abdominal ganglion
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, 1996Co-Authors: T Ushizawa, Toshiki Nagayama, Masakazu TakahataAbstract:Electrical stimulation of mechanosensory afferents innervating hairs on the surface of the exopodite in crayfish Procambarus clarkii (Girard) elicited reciprocal activation of the antagonistic set of Uropod motor neurones. The closer motor neurones were excited while the opener motor neurones were inhibited. This reciprocal pattern of activity in the Uropod motor neurones was also produced by bath application of acetylcholine (ACh) and the cholinergic agonist, carbamylcholine (carbachol). The closing pattern of activity in the Uropod motor neurones produced by sensory stimulation was completely eliminated by bath application of the ACh blocker, d-tubocurarine, though the spontaneous activity of the motor neurones was not affected significantly. Bath application of the acetylcholinesterase inhibitor, neostigmine, increased the amplitude and extended the time course of excitatory postsynaptic potentials (EPSPs) of ascending interneurones elicited by sensory stimulation. These results strongly suggest that synaptic transmission from mechanosensory afferents innervating hairs on the surface of the tailfan is cholinergic.
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sensory control mechanisms of the Uropod equilibrium reflex during walking in the crayfish procambarus clarkii
The Journal of Experimental Biology, 1996Co-Authors: Maki Murayama, Masakazu TakahataAbstract:The temporal characteristics of statocyst and leg proprioceptive inputs to the Uropod motor system were investigated in crayfish using behavioural and electromyographic analyses to elucidate their functional roles in the control of the Uropod steering response under natural conditions. When the animal, which was suspended in the air without a footboard, was actively extending its abdomen, prolonged stimulation of the statocysts by body rolling elicited a maintained asymmetrical configuration of the bilateral Uropods. Prolonged stimulation of the walking legs by footboard tilting with the animal body held in the upright position elicited a transient Uropod response. When the treadmill was tilted while the animal was walking on it in the upright position, the Uropods showed the same transient response. However, when the animal body was rolled, together with the treadmill, while the animal was walking on it, the Uropods showed a transient response which was reversed in direction compared with that observed during body rolling without a footboard. This transient response was abolished by the removal of the statoliths. The results show that the statocysts and leg proprioceptors exert sustained and transient control effects, respectively, on the Uropod motor system during walking. It is also suggested that the Uropod response to body rolling during walking is controlled primarily by leg proprioceptor signals which result from statocyst-induced changes in the leg position.
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terminal projection of descending interneurones controlling Uropod movements of the crayfish procambarus clarkii girard
Zoological Science, 1995Co-Authors: Hisaaki Namba, Toshiki Nagayama, Masakazu TakahataAbstract:Abstract Three types of descending interneurones (type A, B and C) that project into the terminal abdominal ganglion and control the movements of the Uropod have been classified according to their physiological and morphological characters in the crayfish Procambarus clarkii (Girard) using intracellular recording and staining. They were activated by electrical stimulation of a small bundle of isolated fibres in a lateral portion of the ventral nerve cord containing the extension evoking fibres. The type A and B interneurones responded to each stimulus pulse with a spike in one-to-one fashion. The type A interneurones had antagonistic effects on Uropod closer and opener motor neurones, while the type B interneurones coactivated both motor neurone types. The type C interneurones were activated indirectly by electrical stimulation and had antagonistic effects on the Uropod motor neurones. The axons of the three types of interneurones descended in a dorso-lateral regions of the abdominal fifth–sixth connectiv...
Juan M. Serrador - One of the best experts on this subject based on the ideXlab platform.
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Bringing up the rear: defining the roles of the Uropod
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Francisco Sánchez-madrid, Juan M. SerradorAbstract:The Uropod, a protrusion at the rear of amoeboid motile cells such as leukocytes, exemplifies the importance of morphology in cell motility. Although the signalling and structural requirements of Uropod formation are being characterized, a clear understanding of Uropod function is still lacking. Renewed interest in cell shape has been prompted by a recent flood of evidence that indicates that cell polarity is essential for the biology of motile cells. The Uropod, a protrusion at the rear of amoeboid motile cells such as leukocytes, exemplifies the importance of morphology in cell motility. Remodelling of cell shape by Uropod-interfering agents disturbs cell migration. But even though the mechanisms by which Uropods regulate cell migration are beginning to emerge, their functional significance remains enigmatic.
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a juxta membrane amino acid sequence of p selectin glycoprotein ligand 1 is involved in moesin binding and ezrin radixin moesin directed targeting at the trailing edge of migrating lymphocytes
European Journal of Immunology, 2002Co-Authors: Juan M. Serrador, Ana Urzainqui, Jose Luis Alonsolebrero, Roman J Cabrero, Maria C Montoya, Miguel Vicentemanzanares, Maria Yanezmo, Francisco SanchezmadridAbstract:P-selectin glycoprotein ligand 1 (PSGL-1) is an adhesion receptor localized on the tips of microvilli that is involved in the rolling of neutrophils on activated endothelium. We found that PSGL-1 was concentrated at the Uropod of chemokine-stimulated lymphoid cells. Dynamic fluorescence videomicroscopy analyses of migrating lymphocytes demonstrated that PSGL-1 and moesin redistributed towards the cellular Uropod at the trailing edge of these cells, where activated ezrin/radixin/moesin (ERM) proteins were located. An eighteen amino acid sequence in the juxta-membrane region of the PSGL-1 cytoplasmic tail was found to be critical for Uropod targeting and moesin binding. Substitution of S336, S348, and the basic cluster R337K338 by alanines within this region significantly impaired both moesin binding and PSGL-1 polarization. These results underline the role of moesin in the subcellular redistribution of PSGL-1 in lymphoid cells and make evident the importance of specific serine residues within the cytoplasmic tail of PSGL-1 for this process.
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CD43 Interacts With Moesin and Ezrin and Regulates Its Redistribution to the Uropods of T Lymphocytes at the Cell-Cell Contacts
Blood, 1998Co-Authors: Juan M. Serrador, Marta Nieto, José Luis Alonso-lebrero, Miguel A. Pozo, Javier Calvo, Heinz Furthmayr, Reinhard Schwartz-albiez, Francisco Lozano, Roberto González-amaro, Paloma Sánchez-mateosAbstract:Chemokines as well as the signaling through the adhesion molecules intercellular adhesion molecule (ICAM)-3 and CD43 are able to induce in T lymphocytes their switching from a spherical to a polarized motile morphology, with the formation of a Uropod at the rear of the cell. We investigated here the role of CD43 in the regulation of T-cell polarity, CD43-cytoskeletal interactions, and lymphocyte aggregation. Pro-activatory anti-CD43 monoclonal antibody (MoAb) induced polarization of T lymphocytes with redistribution of CD43 to the Uropod and the CCR2 chemokine receptor to the leading edge of the cell. Immunofluorescence analysis showed that all three ezrin-radixin-moesin (ERM) actin-binding proteins localized in the Uropod of both human T lymphoblasts stimulated with anti-CD43 MoAb and tumor-infiltrating T lymphocytes. Radixin localized at the Uropod neck, whereas ezrin and moesin colocalized with CD43 in the Uropod. Biochemical analyses showed that ezrin and moesin coimmunoprecipitated with CD43 in T lymphoblasts. Furthermore, in these cells, the CD43-associated moesin increased after stimulation through CD43. The interaction of moesin and ezrin with CD43 was specifically mediated by the cytoplasmic domain of CD43, as shown by precipitation of both ERM proteins with a GST-fusion protein containing the CD43 cytoplasmic tail. Videomicroscopy analysis of homotypic cell aggregation induced through CD43 showed that cellular Uropods mediate cell-cell contacts and lymphocyte recruitment. Immunofluorescence microscopy performed in parallel showed that Uropods enriched in CD43 and moesin localized at the cell-cell contact areas of cell aggregates. The polarization and homotypic cell aggregation induced through CD43 was prevented by butanedione monoxime, indicating the involvement of myosin cytoskeleton in these phenomena. Altogether, these data indicate that CD43 plays an important regulatory role in remodeling T-cell morphology, likely through its interaction with actin-binding proteins ezrin and moesin. In addition, the redistribution of CD43 to the Uropod region of migrating lymphocytes and during the formation of cell aggregates together with the enhancing effect of anti-CD43 antibodies on lymphocyte cell recruitment suggest that CD43 plays a key role in the regulation of cell-cell interactions during lymphocyte traffic.
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moesin interacts with the cytoplasmic region of intercellular adhesion molecule 3 and is redistributed to the Uropod of t lymphocytes during cell polarization
Journal of Cell Biology, 1997Co-Authors: Juan M. Serrador, Jose Luis Alonsolebrero, Miguel A. Pozo, Javier Calvo, Heinz Furthmayr, Francisco Lozano, Reinhard Schwartzalbiez, Francisco SanchezmadridAbstract:During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a Uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the Uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the Uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the Uropods, whereas moesin decorated the most distal part of the Uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and Uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in Uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.
Janis K. Burkhardt - One of the best experts on this subject based on the ideXlab platform.
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ezrin radixin moesin proteins and flotillins cooperate to promote Uropod formation in t cells
Frontiers in Immunology, 2013Co-Authors: Sibylla Martinelli, Janis K. Burkhardt, Emily J H Chen, Fiona Clarke, Ruth Lyck, Sarah Affentranger, Verena NiggliAbstract:T cell Uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell Uropod. Expression of wild-type ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced Uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and Uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant negative deletion mutant of ezrin, 1-310 ezrin-EGFP, markedly reduced Uropod formation induced by the chemokine SDF-1, T cell front-tail polarity and capping of PSGL-1 and flotillins. Transfection of T cells with wild-type or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1-310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and Uropod formation, indicating that ERM proteins and flotillins cooperate in Uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the Uropod of migrating T cells.
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flotillins and erm proteins function to promote Uropod formation in t cells 44 10
Journal of Immunology, 2010Co-Authors: Janis K. Burkhardt, Verena Niggli, Emily J H Chen, Meredith H ShafferAbstract:Migrating and adherent T cells form a protruding leading edge and a constricted tail-like structure termed the Uropod. The formation of these two structures involves the segregation of specific cytoskeletal elements and cell surface molecules, including proteins that organize cell polarity in other systems. Among the proteins that are segregated to the Uropod are ezrin and moesin, ERM family proteins that organize cell membrane domains by linking cytoplasmic and cytosolic proteins to the actin cytoskeleton. Using conditional ezrin-deficient mice in conjunction with siRNA for moesin, we show that ezrin and moesin are required for Uropod formation. In addition to ERM proteins, we find that the lipid-raft associated actin-binding proteins flotillins/reggies are also associated with the T cell Uropod. Flotillins form a polarized cap even under conditions where a constricted Uropod has not formed. Suppression of flotillin expression inhibits Uropod formation and the polarization of ERM proteins and their binding partners. These data support a model in which flotillins mark a cell surface domain to which ERM proteins are recruited, and ERM proteins then promote formation of a constricted Uropod structure. Since flotillins are not known to interact directly with ERM proteins, intermediary molecules, such as their common binding partner, PSGL-1, may be involved.
Francisco Sanchezmadrid - One of the best experts on this subject based on the ideXlab platform.
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a juxta membrane amino acid sequence of p selectin glycoprotein ligand 1 is involved in moesin binding and ezrin radixin moesin directed targeting at the trailing edge of migrating lymphocytes
European Journal of Immunology, 2002Co-Authors: Juan M. Serrador, Ana Urzainqui, Jose Luis Alonsolebrero, Roman J Cabrero, Maria C Montoya, Miguel Vicentemanzanares, Maria Yanezmo, Francisco SanchezmadridAbstract:P-selectin glycoprotein ligand 1 (PSGL-1) is an adhesion receptor localized on the tips of microvilli that is involved in the rolling of neutrophils on activated endothelium. We found that PSGL-1 was concentrated at the Uropod of chemokine-stimulated lymphoid cells. Dynamic fluorescence videomicroscopy analyses of migrating lymphocytes demonstrated that PSGL-1 and moesin redistributed towards the cellular Uropod at the trailing edge of these cells, where activated ezrin/radixin/moesin (ERM) proteins were located. An eighteen amino acid sequence in the juxta-membrane region of the PSGL-1 cytoplasmic tail was found to be critical for Uropod targeting and moesin binding. Substitution of S336, S348, and the basic cluster R337K338 by alanines within this region significantly impaired both moesin binding and PSGL-1 polarization. These results underline the role of moesin in the subcellular redistribution of PSGL-1 in lymphoid cells and make evident the importance of specific serine residues within the cytoplasmic tail of PSGL-1 for this process.
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lipid rafts mediate biosynthetic transport to the t lymphocyte Uropod subdomain and are necessary for Uropod integrity and function
Blood, 2002Co-Authors: Jaime Millan, Maria C Montoya, Francisco Sanchezmadrid, David Sancho, Miguel A AlonsoAbstract:Polarized migrating T cells possess 2 poles, the Uropod protrusion at the rear and the leading edge at the front, with specific protein composition and function. The influenza virus hemagglutinin (HA) is a prototypical molecule that uses lipid rafts for biosynthetic transport to the apical surface in polarized epithelial Madin-Darby canine kidney (MDCK) cells. In this study, HA was used as a tool to investigate the role of lipid rafts in vectorial protein traffic in polarized T lymphocytes. Results show that newly synthesized HA becomes selectively targeted to the Uropod subdomain in polarized T lymphoblasts. HA incorporates into rafts soon after biosynthesis, suggesting that delivery of HA to the Uropod occurs through a pathway of transport reminiscent of that used for its specific targeting to the apical surface. HA and the adhesion molecules, intercellular adhesion molecule 3 (ICAM-3), CD44, and CD43, 3 endogenous Uropod markers, were detected in surface rafts of T lymphoblasts. Cholesterol, a major component of lipid rafts, was predominantly located in the Uropod. Disruption of lipid raft integrity by cholesterol sequestration produced unclustering of ICAM-3 and the loss of Uropodia and severely impaired processes that require a polarized phenotype such as intercellular aggregation and cell migration. Collectively, these results indicate that lipid rafts constitute a route for selective targeting of proteins to the Uropod and that the rafts are essential for the generation, maintenance, and functionality of T-cell anteroposterior polarity.
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moesin interacts with the cytoplasmic region of intercellular adhesion molecule 3 and is redistributed to the Uropod of t lymphocytes during cell polarization
Journal of Cell Biology, 1997Co-Authors: Juan M. Serrador, Jose Luis Alonsolebrero, Miguel A. Pozo, Javier Calvo, Heinz Furthmayr, Francisco Lozano, Reinhard Schwartzalbiez, Francisco SanchezmadridAbstract:During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a Uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the Uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the Uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the Uropods, whereas moesin decorated the most distal part of the Uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and Uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in Uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.
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chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix involvement of camp signaling pathway
Journal of Cell Biology, 1995Co-Authors: M A Del Pozo, Marta Nieto, Paloma Sanchezmateos, Francisco SanchezmadridAbstract:Leukocyte recruitment is a key step in the inflammatory reaction. Several changes in the cell morphology take place during lymphocyte activation and migration: spheric-shaped resting T cells become polarized during activation, developing a well defined cytoplasmic projection designated as cellular Uropod. We found that the chemotactic and proinflammatory chemokines RANTES, MCP-1, and, to a lower extent, MIP-1 alpha, MIP-1 beta, and IL-8, were able to induce Uropod formation and ICAM-3 redistribution in T lymphoblasts adhered to ICAM-1 or VCAM-1. A similar chemokine-mediated effect was observed during T cells binding to the fibronectin fragments of 38- and 80-kD, that contain the binding sites for the integrins VLA-4 and VLA-5, respectively. The Uropod structure concentrated the ICAM-3 adhesion molecule (a ligand for LFA-1), and emerged to the outer milieu from the area of contact between lymphocyte and protein ligands. In addition, we found that other adhesion molecules such as ICAM-1, CD43, and CD44, also redistributed to the lymphocyte Uropod upon RANTES stimulation, whereas a wide number of other cell surface receptors did not redistribute. Chemokines displayed a selective effect among different T cell subsets; MIP-1 beta had more potent action on CD8+ T cells and tumor infiltrating lymphocytes (TIL), whereas RANTES and MIP-1 alpha targeted selectively CD4+ T cells. We have also examined the involvement of cAMP signaling pathway in Uropod formation. Interestingly, several cAMP agonists were able to induce Uropod formation and ICAM-3 redistribution, whereas H-89, a specific inhibitor of the cAMP-dependent protein kinase, abrogated the chemokine-mediated Uropod formation, thus pointing out a role for cAMP-dependent signaling in the development of this cytoplasmic projection. Since the lymphocyte Uropod induced by chemokines was completely abrogated by Bordetella pertussis toxin, the formation of this membrane projection appears to be dependent on G proteins signaling pathways. In addition, the involvement of myosin-based cytoskeleton in Uropod formation and ICAM-3 redistribution in response to chemokines was suggested by the prevention of this phenomenon with the myosin-disrupting agent butanedione monoxime. Interestingly, this agent also inhibited the ICAM-3-mediated cell aggregation, but not the cell adhesion to substrata. Altogether, these results demonstrate that Uropod formation and adhesion receptor redistribution is a novel function mediated by chemokines; this phenomenon may represent a mechanism that significantly contributes to the recruitment of circulating leukocytes to inflammatory foci.
