Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes

<p>Abstract</p> <p>Background</p> <p>Different types of membrane microdomains (rafts) have been postulated to be present in the rear and front of polarized migrating T-lymphocytes. Disruption of rafts by cholesterol sequestration prevents T-cell polarization and migration. Reggie/flotillin-1 and -2 are two highly homologous proteins that are thought to shape membrane microdomains. We have previously demonstrated the enrichment of flotillins in the uropod of human neutrophils. We have now investigated mechanisms involved in chemokine-induced flotillin reorganization in human T-lymphocytes, and possible roles of flotillins in lymphocyte polarization.</p> <p>Results</p> <p>We studied flotillin reorganization and lateral mobility at the plasma membrane using immunofluorescence staining and FRAP (fluorescence recovery after photobleaching). We show that flotillins redistribute early upon chemokine stimulation, and form very stable caps in the uropods of human peripheral blood T-lymphocytes, colocalizing with the adhesion molecule PSGL-1 and activated ezrin/radixin/moesin (ERM) proteins. Chemokine-induced formation of stable flotillin caps requires integrity and dynamics of the actin cytoskeleton, but is not abolished by inhibitors suppressing Rho-kinase or myosin II activity. Tagged flotillin-2 and flotillin-1 coexpressed in T-lymphocytes, but not singly expressed proteins, colocalize in stable caps at the tips of uropods. Lateral mobility of coexpressed flotillins at the plasma membrane is already partially restricted in the absence of chemokine. Incubation with chemokine results in almost complete immobilization of flotillins. Capping is abolished when wild-type flotillin-1 is coexpressed with a mutant of flotillin-2 (G2A) that is unable to interact with the plasma membrane, or with a deletion mutant of flotillin-2 that lacks a putative actin-binding domain. Wild-type flotillin-2 in contrast forms caps when coexpressed with a mutant of flotillin-1 unable to interact with membranes. Transfection of T-lymphocytes with flotillin-2-G2A reduces cell polarization and uropod recruitment of endogenous flotillin-1 and PSGL-1.</p> <p>Conclusions</p> <p>Our data suggest that stable flotillin cap formation in the rear of polarized T-lymphocytes requires flotillin heterooligomer formation, as well as direct F-actin interactions of flotillin-2 and raft/membrane association of flotillin-2, but not -1. Our data also implicate flotillin-rich actin-dependent membrane microdomains in T-lymphocyte uropod formation.</p

Flotillins Interact with PSGL-1 in Neutrophils and, upon Stimulation, Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

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

Enhanced Activity of Meprin-α, a Pro-Migratory and Pro-Angiogenic Protease, in Colorectal Cancer

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF)- induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer) stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer

Rho-kinase in human neutrophils: a role in signalling for myosin light chain phosphorylation and cell migration

AbstractThe role of a Rho-associated coiled-coil forming kinase in migration of neutrophils has been investigated. Rho-associated coiled-coil forming kinase I was expressed in human neutrophils. Chemotactic peptide led to a Rho-associated coiled-coil forming kinase-dependent increase in phosphorylation of myosin light chain. This was determined with the help of an antibody directed against serine 19-phosphorylated myosin light chain and an inhibitor of Rho-associated coiled-coil forming kinase (Y-27632). Y-27632 suppressed myosin light chain phosphorylation and chemotactic peptide-induced development of cell polarity and locomotion with similar potency (ED50 0.5–1.1 μM). The data strongly suggest that a Rho-associated coiled-coil forming kinase isoform, activated in human neutrophils exposed to chemotactic peptide, is important for motile functions of these cells

Anticipating antiport in P-type ATPases

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(+)-K(+)-ATPase, plasma membrane Ca(2+) ATPase (PMCA) and sarcoplasmic reticulum Ca(2+) ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases

Ezrin/radixin/moesin: Versatile controllers of signaling molecules and of the cortical cytoskeleton

Ezrin, radixin and moesin (ERM) proteins are widely distributed proteins located in the cellular cortex, in microvilli and adherens junctions. They feature an N-terminal membrane binding domain linked by an alpha-helical domain to the C-terminal actin-binding domain. In the dormant state, binding sites in the N-terminal domain are masked by interactions with the C-terminal region. The alpha-helical domain also contributes to masking of binding sites. A specific sequence of signaling events results in dissociation of these intramolecular interactions resulting in ERM activation. ERM molecules have been implicated in mediating actin-membrane linkage and in regulating signaling molecules. They are involved in cell membrane organization, cell migration, phagocytosis and apoptosis, and may also play cell-specific roles in tumor progression. Their precise involvement in these processes has yet to be elucidated

Neutrophil chemotaxis: crucial role of phosphoinositide 3-kinase γ and Rho family GTP-binding proteins

Des neutrophiles représentent le composant principal de l'immunité innée des organismes. Ils sont capables détecter des infections bactériennes, de migrer vers ces dernières afin de détruire les pathogènes envahisseurs. La vaste palette des activités des neutrophiles est contrôlée par un réseau élaboré de cascades de signalisation, qui lient des récepteurs de la surface avec l'intérieur des cellules régulant ainsi les réponses cellulaires au milieu environnant. Des agents chimiotactiques comme le fMLP ou l'interleukin-8 agissent sur les neutrophiles par l'intermédiaire de récepteurs liés aux protéines G trimériques capable d'activer la phosphoinositide 3-kinase de type γ (PI3Kγ), enzyme phosphorylant la position D3 du PIP2 et possédant une activité protéine kinase. Pour élucider le rôle de PI3Kγ dans la signalisation des neutrophiles, nous avons utilisé des souris délétées du gène codant pour la PI3Kγ (PI3Kγ -/-). Nous avons pu montré une diminution des réponses des neutrophiles PI3Kγ-/- à des agents chimiotactiques. En effet, après un "priming" avec du lipopolysaccharide, la production de radicaux oxygénés induite par le fMLP était abolie. Après induction par des agents chimiotactiques, l'adhésion et la polymérisation d'actine des neutrophiles PI3Kγ-/- étaient réduites. Enfin, le chimiotactisme des neutrophiles in vitro et in vivo était diminué, induisant ainsi une protection réduite des souris PI3Kγ -/- contre l'infection bactérienne. Ces données montrent un rôle crucial de la PI3Kγ dans les réponses inflammatoires des neutrophiles et ouvrent des perspectives quant au développement de nouvelles drogues ayant pour cible la PI3Kγ . Les réarrangements du cytosquelette d'actine sont des évènements indispensables pour des activités cellulaires comme le chimiotactisme, l'adhésion, et la phagocytose. Pour caractériser biochimiquement la signalisation conduisant à la polymérisation d'actine, nous avons mis au point un système in vitro utilisant le cytoplasme de neutrophiles humains dans lequel l'ajout de GTPγS (analogue non hydrolysable du GTP) induisait la polymérisation et la formation de réseaux d'actine. Nous avons pu montré que cet effet dépendait de l'activation des petites protéines G de la famille Rho, elle-même sous le contrôle de facteurs d'échanges nucléotidiques (GEF) membranaires, montrant ainsi leur rôle dans le contrôle du cytosquelette d'actine des neutrophiles. De façon surprenante, cette signalisation est indépendante de kinases, excluant l'implication de nombreuses cibles des protéines Rho dans l'initiation de la polymérisation d'actine. Nous avons suggéré que l'effet du GTPγS n'était pas induit par Rac et Cdc42, mais par Rho et une protéine liée à CIP4. L'ensemble de ces données ainsi que la mise au point d'un système in vitro, contribuent à une meilleure compréhension des mécanismes moléculaires impliqués dans la signalisation conduisant au cytosquelette d'actine.Neutrophils represent a key component of organism's innate immunity. They are able to sense bacterial infection, migrate to its source, and destroy the invading pathogen. The vast array of neutrophil activities is controlled by an elaborated net of signal transduction cascades, linking surface receptors with the cell interior and regulating cell responses to the extracellular milieu. Chemoattractants like fMLP or interleukin-8 activate neutrophil receptors coupled to trimeric G proteins. The latter signal to phosphoinositide 3-kinase γ (PI3Kγ), an enzyme phosphorylating D3-position of PIP2 and also possessing a protein kinase activity. To elucidate the role of PI3Kγ in neutrophil signalling, we analyzed neutrophil functions in PI3Kγ knock-out mice. Several responses to chemoattractants were impaired in PI3Kγ -/- neutrophils. After priming with lipopolysaccharide, wild type but not knock-out neutrophils responded to fMLP with a production of oxygen radicals. Chemoattractant-induced cell adhesion and actin polymerization were reduced in PI3Kγ- null neutrophils. Finally, chemotaxis of neutrophils in vitro and in vivo was impaired, resulting in reduced protection of PI3Kγ knock-out mice from infecting bacteria. These data demonstrate a crucial role of PI3Kγ in neutrophil responses in inflammation. They also put forward the enzyme as a promising target for the development of antiinflammatory drugs. Regulated in time and space rearrangements of actin cytoskeleton are crucial to exert such cell activities as chemotaxis, adhesion, and phagocytosis. To characterize biochemically signalling to actin polymerization, we designed a cell-free system from the cytosol of human neutrophils. In this system addition of GTPγS, a non-hydrolyzable analogue of GTP, induced massive actin polymerization and cross-linking. This effect was due to constitutive activation of small Rho family GTP-binding proteins, proving their role in the control of neutrophil actin cytoskeleton. Rho-protein induced actin polymerization was shown to require a plasma membrane-associated guanine nucleotide exchange factor(s). It was also shown to be exerted via a kinase-independent mechanism, excluding a multitude of proposed downstream targets of Rho proteins in the induction of actin polymerization in the neutrophil cytosol. We suggested that Rac and Cdc42 could not be the proteins mediating the effect of GTPγS. In contrast, we found that Rho and a CIP4-binding protein could initiate actin polymerization. These data are an important contribution to the molecular dissecting of cell signalling to actin cytoskeleton, and the established cell-free system will provide more insights into the mechanisms of cell activation

Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells

Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis