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.

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.

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.

Chemokines guide lymphocytes from blood to secondary lymphoid organs by triggering integrin-dependent firm adhesion under vascular flow and directed migration of T and B lymphocytes within lymphoid tissue. Here, we analyze the roles of DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, and phosphoinositide-3-kinase (PI3K) during lymphocyte recirculation. DOCK2 mediated efficient lymphocyte migration in a largely PI3K-independent manner, although a minor, PI3K-dependent pathway for migration was observed in wild-type and DOCK2-deficient lymphocytes. In T cells, this residual migration depended mainly on PI3Kgamma, whereas other PI3K isoforms were implicated in B cells. In vitro adhesion assays and intravital microscopy of lymphoid organ vasculature uncovered an unexpected defect in integrin activation in DOCK2-/- B cells, whereas lack of DOCK2 did not affect chemokine-triggered integrin activation in T cells. DOCK2 and PI3Kgamma thus play distinct roles during T and B cell integrin activation and migration.

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 b1 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.

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 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.

The mammalian epiblast is formed by pluripotent cells able to differentiate into all tissues of the new individual. In their progression to differentiation, epiblast cells and their in vitro counterparts, embryonic stem cells (ESCs), transit from naive pluripotency through a differentiation-primed pluripotent state. During these events, epiblast cells and ESCs are prone to death, driven by competition between Myc-high cells (winners) and Myc-low cells (losers). Using live tracking of Myc levels, we show that Myc-high ESCs approach the naive pluripotency state, whereas Myc-low ESCs are closer to the differentiation-primed state. In ESC colonies, naive cells eliminate differentiating cells by cell competition, which is determined by a limitation in the time losers are able to survive persistent contact with winners. In the mouse embryo, cell competition promotes pluripotency maintenance by elimination of primed lineages before gastrulation. The mechanism described here is relevant to mammalian embryo development and induced pluripotency.