María C. Montoya

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 α-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.

Recruitment of Nck by CD3ϵ Reveals a Ligand-Induced Conformational Change Essential for T Cell Receptor Signaling and Synapse Formation

How membrane receptors initiate signal transduction upon ligand binding is a matter of intense scrutiny. The T cell receptor complex (TCR-CD3) is composed of TCR alpha/beta ligand binding subunits bound to the CD3 subunits responsible for signal transduction. Although it has long been speculated that TCR-CD3 may undergo a conformational change, confirmation is still lacking. We present strong evidence that ligand engagement of TCR-CD3 induces a conformational change that exposes a proline-rich sequence in CD3 epsilon and results in recruitment of the adaptor protein Nck. This occurs earlier than and independently of tyrosine kinase activation. Finally, by interfering with Nck-CD3 epsilon association in vivo, we demonstrate that TCR-CD3 recruitment of Nck is critical for maturation of the immune synapse and for T cell activation.

MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway

MT1‐matrix metalloproteinase (MT1‐MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1‐MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1‐MMP occurs during MDA‐MB‐231 adenocarcinoma cell migration into collagen type I three‐dimensional matrices. Polarized trafficking of MT1‐MMP is triggered by β1 integrin‐mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1‐MMP within VSV‐G/Rab8‐positive vesicles, but not in Rab11/Tf/TfRc‐positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1‐MMP exocytic traffic, collagen degradation and invasion, whereas Rab8‐ but not Rab11‐knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1‐MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1‐MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.

Role of ICAM-3 in the initial interaction of T lymphocytes and APCs

Antigen-independent adhesive interactions between T lymphocytes and antigen-presenting cells (APCs) are essential for scanning for specific antigens on the APC surface and for initiating the immune response. Here we show, through time-lapse imaging of live cells, that the intercellular adhesion molecule 3 (ICAM-3, also known as CD50) is clustered specifically at the region of the T lymphocyte surface that initiates contact with APCs. We describe the role of ICAM-3 in T cell–APC conjugate formation before antigen recognition, in early intracellular signaling and in cytoskeletal rearrangement. Our data indicate that ICAM-3 is important in the initial scanning of the APC surface by T cells and, therefore, in generating the immune response.

The chemokine SDF‐1α triggers a chemotactic response and induces cell polarization in human B lymphocytes

We studied the expression and possible functional role of chemokine receptors CXCR3, CXCR4 and CCR5 in normal human B lymphocytes. B cells from both peripheral blood and tonsils expressed high levels of CXCR4 but not the other chemokine receptors tested. CXCR4 ligand, stromal cell‐derived factor (SDF)‐1α, elicited a potent chemotactic response and induced a polarized motile phenotype in B cells, resulting in redistribution of the adhesion molecule ICAM‐3 to a posterior appendage of the cell, termed uropod, and of CXCR4 receptor to the leading edge of migrating B cells. Time‐lapse videomicroscopy studies revealed that SDF‐1α‐treated cells recruited additional bystander B cells through the uropod. SDF‐1α induced levels of cellular recruitment comparable to those elicited by polarization‐inducing anti‐ICAM‐3 monoclonal antibody, in an LFA‐1/ICAM‐1, −3‐dependent fashion. Moreover, this chemokine increased intracellular Ca2+ levels in B lymphocytes, and induced a rapid CXCR4 receptor down‐regulation on the cell surface membrane. These results provide new insight into the important biological role of SDF‐1α in physiological processes in which B cells participate, and suggest a key role for chemokines in normal B cell trafficking and recirculation.

Screening out irrelevant cell-based models of disease.

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment

The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated ζ chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.

Cell adhesion and polarity during immune interactions

Summary: Intercellular interactions are critical for a coordinated function of different cell types involved in the immune response. Here we review the cellular and molecular events occurring during cell–cell immune contacts. Cognate naïve CD4+ T lymphocyte‐dendritic cell (DC) and primed T cell–antigen‐presenting B lymphocyte interactions are discussed. The engagement of cytotoxic T lymphocytes (CTL) or natural killer cells (NK) with their targets is analyzed and compared to the process of T cell–antigen‐presenting cell (APC) conjugate formation. The immunological synapse, a complex cluster of molecules organized at the contact area of cell conjugates, exhibits common features but shows some differences depending on cell types involved. Cellular interactions occur in sequential stages that involve dramatic changes in cell polarity and dynamic redistribution of cell membrane receptors. The role of membrane microdomains, adaptor molecules and the cytoskeleton in the regulation of the molecular reorganization at cell–cell contacts is also discussed.

MT1-MMP collagenolytic activity is regulated through association with tetraspanin CD151 in primary endothelial cells

MT1-MMP plays a key role in endothelial function, as underscored by the angiogenic defects found in MT1-MMP deficient mice. We have studied the molecular interactions that underlie the functional regulation of MT1-MMP. At lateral endothelial cell junctions, MT1-MMP colocalizes with tetraspanin CD151 (Tspan 24) and its associated partner alpha3beta1 integrin. Biochemical and FRET analyses show that MT1-MMP, through its hemopexin domain, associates tightly with CD151, thus forming alpha3beta1 integrin/CD151/MT1-MMP ternary complexes. siRNA knockdown of HUVEC CD151 expression enhanced MT1-MMP-mediated activation of MMP2, and the same activation was seen in ex vivo lung endothelial cells isolated from CD151-deficient mice. However, analysis of collagen degradation in these experimental models revealed a diminished MT1-MMP enzymatic activity in confined areas around the cell periphery. CD151 knockdown affected both MT1-MMP subcellular localization and its inclusion into detergent-resistant membrane domains, and prevented biochemical association of the metalloproteinase with the integrin alpha3beta1. These data provide evidence for a novel regulatory role of tetraspanin microdomains on the collagenolytic activity of MT1-MMP and indicate that CD151 is a key regulator of MT1-MMP in endothelial homeostasis.

Vitrification versus slow freezing of oocytes: effects on morphologic appearance, meiotic spindle configuration, and DNA damage

To assess possible effects on subcellular organization after cryopreservation, we compared vitrified and slowly frozen oocytes in terms of their post-warm/thaw morphology, meiotic spindle configuration, and DNA integrity. DNA integrity of cryopreserved oocytes was not altered after the procedures, but vitrification was more effective than slow cooling, as shown by higher survival rate and spindle assessment despite a higher misalignment between meiotic spindle and polar body.