Biomedical subjects

Olga Barreiro

Publications and source records attributed to Olga Barreiro.

10 recordsLinked to original sources

PubMed2006

Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion.

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.

Antigens, CD↗

PubMed2006

Lymphocyte chemotaxis is regulated by histone deacetylase 6, independently of its deacetylase activity.

In this work, the role of HDAC6, a type II histone deacetylase with tubulin deacetylase activity, in lymphocyte polarity, motility, and transmigration was explored. HDAC6 was localized at dynamic subcellular structures as leading lamellipodia and the uropod in migrating T-cells. However, HDAC6 activity did not appear to be involved in the polarity of migrating lymphocytes. Overexpression of HDAC6 in freshly isolated lymphocytes and T-cell lines increased the lymphocyte migration mediated by chemokines and their transendothelial migration under shear flow. Accordingly, the knockdown of HDAC6 expression in T-cells diminished their chemotactic capability. Additional experiments with HDAC6 inhibitors (trichostatin, tubacin), other structural related molecules (niltubacin, MAZ-1391), and HDAC6 dead mutants showed that the deacetylase activity of HDAC6 was not involved in the modulatory effect of this molecule on cell migration. Our results indicate that HDAC6 has an important role in the chemotaxis of T-lymphocytes, which is independent of its tubulin deacetylase activity.

Acetylation↗

PubMed2005

Role of tetraspanins CD9 and CD151 in primary melanocyte motility.

Tetraspanins CD9 and CD151 have been implicated in cellular motility and intercellular adhesion in several cellular types. Here, we have studied the subcellular localization and the functional role of these molecules in primary melanocytes. We found that endogenous tetraspanins preferentially clustered in areas of melanocyte homotypic intercellular contacts and at the tips of dendrites. These observations were further confirmed using time-lapse fluorescence confocal microscopy of melanocytes transfected with CD9- and CD151-GFP (green fluorescent protein) constructs, suggesting an involvement of these proteins in cellular contacts and migration. Cell adhesion and migration assays performed using blocking monoclonal antibodies against CD9 and CD151 showed no significant effect on cell-extracellular matrix adhesion, whereas the migration of melanocytes was significantly enhanced. The regulation of the migratory capacity of melanocytes by CD9 and CD151 was further confirmed knocking down the endogenous expression of these tetraspanins with small interference RNA oligonucleotides. Therefore, tetraspanin molecules are localized at motile structures in primary human melanocytes regulating the migratory capacity of these cells.

Antigens, CD↗

PubMed2004

Endothelial tetraspanin microdomains regulate leukocyte firm adhesion during extravasation.

Tetraspanins associate with several transmembrane proteins forming microdomains involved in intercellular adhesion and migration. Here, we show that endothelial tetraspanins relocalize to the contact site with transmigrating leukocytes and associate laterally with both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Alteration of endothelial tetraspanin microdomains by CD9-large extracellular loop (LEL)-glutathione S-transferase (GST) peptides or CD9/CD151 siRNA oligonucleotides interfered with ICAM-1 and VCAM-1 function, preventing lymphocyte transendothelial migration and increasing lymphocyte detachment under shear flow. Heterotypic intercellular adhesion mediated by VCAM-1 or ICAM-1 was augmented when expressed exogenously in the appropriate tetraspanin environment. Therefore, tetraspanin microdomains have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration.

Antigens, CD↗

PubMed2004

Interactive protrusive structures during leukocyte adhesion and transendothelial migration.

Leukocyte transendothelial migration during homing and inflammation requires drastic cell morphological changes, involving cytoskeletal-directed clustering of adhesion receptors in specialized protrusive membrane structures in leukocytes and endothelial cells. Extravasation is an active process not only for leukocytes but also for endothelial cells, which promote the rapid and efficient entry of leukocytes to the target tissues, without disturbing the integrity of the endothelial barrier. Herein, we have revised the specialized protrusive structures (microvilli, endothelial docking structures, leukocyte lamellipodia and uropod) involved in the different stages of leukocyte extravasation. The adhesion receptor redistribution, cytoskeletal remodelling and intracellular signaling events that participate in this phenomenon are also discussed.

Animals↗

PubMed2003

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes.

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.

Actins↗

PubMed2002

Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes.

The binding of chemokines to their receptors guides lymphocyte migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by the actomyosin complex of the cell has not been elucidated. Using biochemical approaches and mass spectrometry analysis, we found an association between the C-termini of CXCR4 and CCR5 and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As expected, myosin L chain was also associated with CXCR4. Confocal microscopy analysis showed that CXCR4 and motor protein nonmuscle myosin H chain-IIA colocalize at the leading edge of migrating T lymphocytes, together with filamentous actin and myosin L chain. These results provide the first evidence of a biochemical association between chemokine receptors and motor proteins, a mechanosignaling mechanism that may have a key role in lymphocyte migration.

Actins↗

PubMed2002

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes.

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as alpha-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Actins↗

PubMed2002

A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting.

Intercellular adhesion molecule 3 (ICAM-3) is a leukocyte-specific receptor involved in primary immune responses. We have investigated the interaction between ICAM-3 and ezrin/radixin/moesin (ERM) proteins and its role in LFA-1-induced cell-cell interactions and membrane positioning of ICAM-3 in polarized migrating lymphocytes. Protein-protein binding assays demonstrated a phosphatidylinositol 4,5-bisphosphate-induced association between ICAM-3 and the amino-terminal domain of ERM proteins. This interaction was not essential for the binding of ICAM-3 to LFA-1. Dynamic fluorescence videomicroscopy studies of cells demonstrated that moesin and ICAM-3 coordinately redistribute on the plasma membrane during lymphocyte migration. Furthermore, overexpression of the amino-terminal domain of moesin, which lacks the consensus moesin actin-binding site, caused the subcellular mislocalization of ICAM-3. A CD4 chimerical protein containing the cytoplasmic tail of ICAM-3 was targeted to the trailing edge. Point mutation of Ser(487), Ser(489), and Ser(496) to alanine in the juxtamembrane region of ICAM-3 significantly impaired both ERM binding and polarization of ICAM-3. ERM-directed polarization of ICAM-3 was also impaired by phosphorylation-like mutation of Ser(487) and Ser(489), but not of Ser(496). Our results underscore the key role of specific serine residues within the cytoplasmic region of ICAM-3 for its ERM-directed positioning at the trailing edge of motile lymphocytes.

Alanine↗

PubMed2002

Effect of pentoxifylline on polarization and migration of human leukocytes.

Leukocyte polarization has a key role in the induction and effector phases of immune response. We assessed the effect of pentoxifylline (PTX) on the polarization and migration of human lymphocytes and neutrophils. A dose-dependent, inhibitory effect on the polarization of lymphoid cells induced by chemokines or IL-15 was found. In addition, PTX interfered with the chemotaxis of peripheral blood T cells and T lymphoblasts. A similar effect was observed on the transendothelial migration of these cells. In addition, the polarization of neutrophils, its adherence to endothelium, and their transendothelial migration, induced by different stimuli, were inhibited by PTX. By contrast, this drug had only a mild effect on endothelial cells and a partial inhibition on the induction of ICAM-1 expression by TNF-alpha. The inhibitory effect of PTX on leukocyte polarization and extravasation may contribute significantly to the anti-inflammatory and immunoregulatory activity of this drug.

Cell Adhesion↗