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Notch signalling has a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development; here two adhesion molecules, Jam1a and Jam2a, are shown to be essential for the contact between precursors of HSCs and the somite during embryonic migration, and the Jam1a–Jam2a interaction is shown to be needed to transmit the Notch signal and produce HSCs. Haematopoietic stem cells in the embryo Two papers published in this issue of Nature demonstrate the involvement of somites — paired masses of mesoderm cells that form along the anterior–posterior axis of the embryo — in the generation of haematopoietic stem cells (HSCs) during vertebrate development. Phong Dang Nguyen et al . identify a previously unknown somite compartment, called the endotome, which contributes to the formation of the embryonic dorsal aorta by providing endothelial progenitors. The formation of the endotome is regulated by the activity of meox1 , a homeobox-containing transcription factor. Isao Kobayashi et al . report that the precursors of HSCs make direct contact with the somite during their embryonic migration and that the interaction is needed to receive the necessary Notch signal. They identify two adhesion molecules that mediate the contact: Jam1a, which is expressed by HSC precursors, and Jam2a, which is expressed by the somite. Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development 1 , 2 , 3 and requires intimate contact between signal-emitting and signal-receiving cells, although little is known regarding when, where and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification 4 . It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a (also known as f11r ) and migrate axially across the ventral somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a–Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses 1 , 2 , forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly—which was not detected in undecorated virions—is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus 3 ; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae . Cryo-electron microscopy structures of feline calicivirus and its cellular receptor show that twelve copies of the minor capsid protein VP2 form a portal-like assembly arranged about a pore in the capsid shell.

Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell–cell contacts with enrichment at the tight junctions. Its role during cell–cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3β1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3β1 integrin and regulate α3β1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3β1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3β1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3β1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.

Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) regulate important processes such as cell proliferation, differentiation and morphogenesis. This activity is primarily due to their ability to initiate intracellular signaling cascades at cell–cell contact sites. Junctional adhesion molecule-A (JAM-A) is an IgSF-CAM with a short cytoplasmic tail that has no catalytic activity. Nevertheless, JAM-A is involved in a variety of biological processes. The functional diversity of JAM-A resides to a large part in a C-terminal PDZ domain binding motif which directly interacts with nine different PDZ domain-containing proteins. The molecular promiscuity of its PDZ domain motif allows JAM-A to recruit protein scaffolds to specific sites of cell–cell adhesion and to assemble signaling complexes at those sites. Here, we review the molecular characteristics of JAM-A, including its dimerization, its interaction with scaffolding proteins, and the phosphorylation of its cytoplasmic domain, and we describe how these characteristics translate into diverse biological activities.

There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.

Patients with risks of ischemic injury, e.g. during circulatory arrest in cardiac surgery, or after resuscitation are subjected to therapeutic hypothermia. For aortic surgery, the body is traditionally cooled down to 18 °C and then rewarmed to body temperature. The role of hypothermia and the subsequent rewarming process on leukocyte-endothelial interactions and expression of junctional-adhesion-molecules is not clarified yet. Thus, we investigated in an in-vitro model the influence of temperature modulation during activation and transendothelial migration of leukocytes through human endothelial cells. Additionally, we investigated the expression of JAMs in the rewarming phase. Exposure to low temperatures alone during transmigration scarcely affects leukocyte extravasation, whereas hypothermia during treatment and transendothelial migration improves leukocyte-endothelial interactions. Rewarming causes a significant up-regulation of transmigration with falling temperatures. JAM-A is significantly modulated during rewarming. Our data suggest that transendothelial migration of leukocytes is not only modulated by cell-activation itself. Activation temperatures and the rewarming process are essential. Continued hypothermia significantly inhibits transendothelial migration, whereas the rewarming process enhances transmigration strongly. The expression of JAMs, especially JAM-A, is strongly modulated during the rewarming process. Endothelial protection prior to warm reperfusion and mild hypothermic conditions reducing the difference between hypothermia and rewarming temperatures should be considered.

The mammalian JAM family is composed of three cell surface receptors. Interactions between the proteins have well-characterised roles in inflammation and tight junction formation, but little is known about their function in early development. Recently, we identified a role for jamb and jamc in zebrafish myocyte fusion. Genome duplication in the teleost lineage raised the possibility that additional JAM family paralogues may also function in muscle development. To address this, we searched the zebrafish genome to identify potential paralogues and confirmed their homology, bringing the total number of zebrafish jam family members to six. We then compared the physical binding properties of each paralogue by surface plasmon resonance and determined the gene expression patterns of all zebrafish jam genes at different stages of development. Our results suggest a significant sub-functionalisation of JAM-B and JAM-C orthologues with respect to binding strength (but not specificity) and gene expression. The paralogous genes, jamb2 and jamc2, were not detected in the somites or myotome of wild-type embryos. We conclude that it is unlikely that the paralogues have a function in primary myogenesis.

Recent studies have shown that transmembrane‐type tight junction proteins are upregulated in various cancers compared with their levels in normal tissues and are involved in cancer progression, suggesting that they are potential therapeutic targets. Here, we demonstrated the expression profile and a novel role of junctional adhesion molecule‐A (JAM‐A) in breast cancer. Immunohistochemistry of surgical specimens showed that JAM‐A was highly expressed from carcinoma in situ lesions, as in other adenocarcinomas, with higher expression in invasive carcinomas. High expression of JAM‐A contributed to malignant aspects such as lymph node metastasis and lymphatic involvement positivity. In breast cancer cells, JAM‐A expression status affects malignant potentials including proliferation and migration. Multilayered proteomics revealed that JAM‐A interacts with the amino acid transporter LAT1 in breast cancer cells. JAM‐A regulates the expression of LAT1 and interacts with it on the whole cell membrane, leading to enhanced amino acid uptake to promote tumor growth. Double high expression of JAM‐A and LAT1 predicts poor prognosis in patients with breast cancer. Of note, an antibody against an extracellular domain of JAM‐A suppressed the proliferation of breast cancer cells. Our findings indicate the possibility of JAM‐A‐targeted therapy ideally combined with LAT1‐targeted therapy as a new therapeutic strategy against breast cancer. We uncovered junctional adhesion molecule‐A (JAM‐A), one of the transmembrane‐type tight junction proteins (TJPs), promoted breast cancer progression via regulation of amino acid transporter LAT1 using multilayered proteomics. Our findings provide a new insight into a therapeutic strategy, JAM‐A‐targeted therapy ideally combined with LAT1‐targeted therapy, against breast cancer. Our multilayered proteomics approach can also apply to investigating the roles of other transmembrane‐type TJPs in various malignancies.

This study aimed to elucidate the roles of junctional adhesion molecule 2 (JAM‐2), collagen I and matrix metalloproteinase 2 (MMP‐2) in the pathogenesis of pelvic organ prolapse (POP) and explore their potential as diagnostic markers. We examined 82 POP patients and 64 controls using enzyme‐linked immunosorbent assay (ELISA) and quantitative Polymerase Chain Reaction (qPCR) to analyse protein and gene expression levels of JAM‐2, Collagen I and MMP‐2. Receiver operating characteristic (ROC) analysis evaluated their diagnostic efficacy, with correlation analyses linking molecular markers to POP severity based on POP‐Q grades. Our study found no significant differences in age, BMI and vaginal parity between POP patients and controls. Molecular analyses revealed significant alterations in the expression levels of JAM‐2, Collagen I and MMP‐2 in POP patients. Specifically, there was a marked decrease in JAM‐2 and collagen I levels, accompanied by an increase in MMP‐2 expression, indicating a disruption in the balance between tissue synthesis and degradation. ROC analysis demonstrated the significant discriminative power of these markers, with substantial area under the curve (AUC) values for diagnosing POP. Correlation analysis further showed a significant association between the expression of JAM‐2, Collagen I and MMP‐2 and the clinical severity of POP, as indicated by POP‐Q grades. Our findings revealed the significant changes in the expression of JAM‐2, Collagen I and MMP‐2 that may contribute to the POP pathogenesis. The diagnostic potential of these markers was substantiated, suggesting their utility in developing noninvasive diagnostic tools for POP.

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM-C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.