Jaime Millán

MAL, a novel integral membrane protein of human T lymphocytes, associates with glycosylphosphatidylinositol-anchored proteins and Src-like tyrosine kinases.

A large fraction of glycosylphosphatidylinositol (GPI)‐anchored proteins and Src‐like kinases are confined to glycolipid‐enriched membrane (GEM) microdomains. The particular membrane topology of GPI‐anchored proteins has led to the postulation of the existence of integral membrane proteins linking extracellular stimuli with cytosolic machinery for endocytosis and signaling. The human MAL cDNA was identified during a search for novel genes differentially expressed during T cell development, and encodes a multispanning membrane protein displaying lipid‐like properties. To address the biochemical characterization of endogenous MAL and to analyze its possible association with other proteins, we have generated a monoclonal antibody (mAb) specific to the MAL molecule. Using this mAb, we have identified MAL in GEM microdomains of both the HPB‐ALL T cell line and human peripheral blood lymphocytes. Co‐immunoprecipitation experiments with antibodies to the MAL molecule or to the GPI‐anchored CD59 antigen indicated specific association of MAL with GPI‐anchored proteins and Src‐like tyrosine kinases. In addition, both MAL and the Src‐like kinase Lck were identified in GEM obtained from an endosomal‐enriched membrane fraction. These features of MAL closely match some of the properties expected for the hypothetical integral membrane linker proteins acting in specialized GEM‐mediated functions.

An essential role for the MAL protein in targeting Lck to the plasma membrane of human T lymphocytes.

The MAL protein is an essential component of the specialized machinery for apical targeting in epithelial cells. The src family kinase Lck plays a pivotal role in T cell signaling. We show that MAL is required in T cells for efficient expression of Lck at the plasma membrane and activation of IL-2 transcription. To investigate the mechanism by which MAL regulates Lck targeting, we analyzed the dynamics of Lck and found that it travels to the plasma membrane in specific transport carriers containing MAL. Coimmunoprecipitation experiments indicated an association of MAL with Lck. Both carrier formation and partitioning of Lck into detergent-insoluble membranes were ablated in the absence of MAL. Polarization of T cell receptor for antigen (TCR) and microtubule-organizing center to immunological synapse (IS) were also defective. Although partial correction of the latter defects was possible by forced expression of Lck at the plasma membrane, their complete correction, formation of transport vesicles, partitioning of Lck, and restoration of signaling pathways, which are required for IL-2 transcription up-regulation, were achieved by exogenous expression of MAL. We concluded that MAL is required for recruitment of Lck to specialized membranes and formation of specific transport carriers for Lck targeting. This novel transport pathway is crucial for TCR-mediated signaling and IS assembly.

TNF-induced endothelial barrier disruption: beyond actin and Rho

The decrease of endothelial barrier function is central to the long-term inflammatory response. A pathological alteration of the ability of endothelial cells to modulate the passage of cells and solutes across the vessel underlies the development of inflammatory diseases such as atherosclerosis and multiple sclerosis. The inflammatory cytokine tumour necrosis factor (TNF) mediates changes in the barrier properties of the endothelium. TNF activates different Rho GTPases, increases filamentous actin and remodels endothelial cell morphology. However, inhibition of actin-mediated remodelling is insufficient to prevent endothelial barrier disruption in response to TNF, suggesting that additional molecular mechanisms are involved. Here we discuss, first, the pivotal role of Rac-mediated generation of reactive oxygen species (ROS) to regulate the integrity of endothelial cell-cell junctions and, second, the ability of endothelial adhesion receptors such as ICAM-1, VCAM-1 and PECAM-1, involved in leukocyte transendothelial migration, to control endothelial permeability to small molecules, often through ROS generation. These adhesion receptors regulate endothelial barrier function in ways both dependent on and independent of their engagement by immune cells, and orchestrate the crosstalk between leukocyte transendothelial migration and endothelial permeability during inflammation.

Recruitment of Transferrin Receptor to Immunological Synapse in Response to TCR Engagement

T cell receptor engagement by an APC induces the formation of a highly organized complex of surface receptors and intracellular signaling molecules, known as the immunological synapse, at the site of cell-cell contact. The transferrin receptor (TfR, CD71) is normally present in the plasma membrane and recycling endosomes. In this study, we show that, although the TfR is typically absent from lipid rafts at steady state, stimulation with a mitogenic mixture of anti-CD3 Abs of human Jurkat T cells leads to a rapid compartmentalization of the TfR into lipid rafts accompanying that of CD3ε and activated Lck. This change occurs very rapidly and is accompanied by an increase in the surface expression of the TfR, probably by translocation from an internal endosomal pool. TfR recruitment to lipid rafts was also observed in primary T cells treated with mitogenic anti-CD3 Abs and in Jurkat T cell-APC conjugates. The use of beads coated with Abs indicates that the surface and endosomal TfR pools redistribute to the contact site region in response to engagement of CD28 and CD3. In T cell-APC conjugates, the T cell TfR endosomal pool relocates beneath the contact site, whereas surface TfR localizes to the peripheral ring of the immunological synapse. In the presence of specific anti-TfR Abs, the total number of T cell-APC contacts and the percentage of conjugates with CD3 and Lck translocated to the contact site were reduced. Our results therefore suggest the involvement of the TfR in the formation of the immunological synapse.

Rac activation by the T-cell receptor inhibits T cell migration.

Background T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR) triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known. Methodology/Principal Findings Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin. Conclusions/Significance We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.

Segregation of co-stimulatory components into specific T cell surface lipid rafts

The glycosylphosphatidylinositol (GPI)‐anchored protein CD59 and the ganglioside GM1 are present on lipid rafts that can be isolated in a detergent‐insoluble membrane (DIM) fraction. TCR engagement promotes integration of components of the TCRu2009/u2009CD3 signaling machinery into DIM. As DIM are isolated as a heterogeneous mixture of coalescent membranes, it is uncertain whether the cofractionation of GPI‐anchored proteins and GM1 reflects the existence of an association between these molecules within the same lipid rafts in the cell. We have studied the surface distribution of the co‐stimulatory CD59 and GM1 molecules and their role in the recruitment of components of the TCR signaling machinery in DIM. Although both CD59 and GM1 are present in rafts, these molecules occur in a steady state, mainly clustered in different membrane subdomains. Multimerization of either molecule did not induce cocapping or co‐internalization of the other. Aggregation of GM1, CD59 or TCRu2009/u2009CD3 increased tyrosine phosphorylation but only in the latter case was a significant increase observed in both tyrosine phosphorylation and recruitment of elements of the signaling machinery in DIM. Our results show the existence of specific co‐stimulatory membrane microdomains that require a direct TCRu2009/u2009CD3 engagement for efficient recruitment of signaling machinery into rafts.

Endothelial membrane reorganization during leukocyte extravasation

Leukocyte trafficking from the bloodstream to inflamed tissues across the endothelial barrier is an essential response in innate immunity. Leukocyte adhesion, locomotion, and diapedesis induce signaling in endothelial cells and this is accompanied by a profound reorganization of the endothelial cell surfaces that is only starting to be unveiled. Here we review the current knowledge on the leukocyte-mediated alterations of endothelial membrane dynamics and their role in promoting leukocyte extravasation. The formation of protein- and lipid-mediated cell adhesion nanodomains at the endothelial apical surface, the extension of micrometric apical membrane docking structures, which are derived from microvilli and embrace adhered leukocytes, as well as the vesicle-trafficking pathways that are required for efficient leukocyte diapedesis, are discussed. The coordination between these different endothelial membrane-remodeling events probably provides the road map for transmigrating leukocytes to find exit points in the vessel wall, in a context of severe mechanical and inflammatory stress. A better understanding of how vascular endothelial cells respond to immune cell adhesion should enable new therapeutic strategies to be developed that can abrogate uncontrolled leukocyte extravasation in inflammatory diseases.

Microparticles in multiple sclerosis and clinically isolated syndrome: effect on endothelial barrier function.

BackgroundCell-derived microparticles are secreted in response to cell damage or dysfunction. Endothelial and platelet dysfunction are thought to contribute to the development of multiple sclerosis (MS). Our aim here is, first, to compare the presence of microparticles of endothelial and platelet origin in plasma from patients with different clinical forms of MS and with clinically isolated syndrome. Second, to investigate the effect of microparticles on endothelial barrier function.ResultsPlatelet-poor plasma from 95 patients (12 with clinically isolated syndrome, 51 relapsing-remitting, 23 secondary progressive, 9 primary progressive) and 49 healthy controls were analyzed for the presence of platelet-derived and endothelium-derived microparticles by flow cytometry. The plasma concentration of platelet-derived and endothelium-derived microparticles increased in all clinical forms of MS and in clinically isolated syndrome versus controls. The response of endothelial barriers to purified microparticles was measured by electric cell-substrate impedance sensing. Microparticles from relapsing-remitting MS patients induced, at equivalent concentrations, a stronger disruption of endothelial barriers than those from healthy donors or from patients with clinically isolated syndrome. MS microparticles acted synergistically with the inflammatory mediator thrombin to disrupt the endothelial barrier function.ConclusionsPlasma microparticles should be considered not only as markers of early stages of MS, but also as pathological factors with the potential to increase endothelial permeability and leukocyte infiltration.

Crosstalk Between Reticular Adherens Junctions and Platelet Endothelial Cell Adhesion Molecule-1 Regulates Endothelial Barrier Function

Objective—Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. Methods and Results—In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins &bgr;-catenin and p120-catenin. &bgr;-catenin binds to &agr;-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-&agr; increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-&agr;. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-&agr;–induced alterations of barrier function, including increased &bgr;-catenin phosphorylation, without altering the actomyosin cytoskeleton. Conclusion—We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-&agr;.

Developmental regulation of apical endocytosis controls epithelial patterning in vertebrate tubular organs

Epithelial organs develop through tightly coordinated events of cell proliferation and differentiation in which endocytosis plays a major role. Despite recent advances, how endocytosis regulates the development of vertebrate organs is still unknown. Here we describe a mechanism that facilitates the apical availability of endosomal SNARE receptors for epithelial morphogenesis through the developmental upregulation of plasmolipin (pllp) in a highly endocytic segment of the zebrafish posterior midgut. The protein PLLP (Pllp in fish) recruits the clathrin adaptor EpsinR to sort the SNARE machinery of the endolysosomal pathway into the subapical compartment, which is a switch for polarized endocytosis. Furthermore, PLLP expression induces apical Crumbs internalization and the activation of the Notch signalling pathway, both crucial steps in the acquisition of cell polarity and differentiation of epithelial cells. We thus postulate that differential apical endosomal SNARE sorting is a mechanism that regulates epithelial patterning.