PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

Junctional Adhesion Molecules (JAMs): Cell Adhesion Receptors With Pleiotropic Functions in Cell Physiology and Development.

Junctional adhesion molecules (JAM)-A, -B and -C are cell-cell adhesion molecules of the immunoglobulin superfamily which are expressed by a variety of tissues, both during development and in the adult organism. Through their extracellular domains, they interact with other adhesion receptors on opposing cells. Through their cytoplasmic domains, they interact with PDZ domain-containing scaffolding and signaling proteins. In combination, these two properties regulate the assembly of signaling complexes at specific sites of cell-cell adhesion. The multitude of molecular interactions has enabled JAMs to adopt distinct cellular functions such as the regulation of cell-cell contact formation, cell migration, or mitotic spindle orientation. Not surprisingly, JAMs regulate diverse processes such as epithelial and endothelial barrier formation, hemostasis, angiogenesis, hematopoiesis, germ cell development, and the development of the central and peripheral nervous system. This review summarizes the recent progress in the understanding of JAMs, including their characteristic structural features, their molecular interactions, their cellular functions, and their contribution to a multitude of processes during vertebrate development and homeostasis.

JAM-A interacts with α3ß1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells.

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 α3ß1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3ß1 integrin and regulate α3ß1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3ß1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3ß1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3ß1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.

TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function.

OBJECTIVE: We explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability. DESIGN: Experiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied. RESULTS: In vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice. CONCLUSION: The BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.

Regulation of cell polarity by cell adhesion receptors.

The ability of cells to polarize is an intrinsic property of almost all cells and is required for the devlopment of most multicellular organisms. To develop cell polarity, cells integrate various signals derived from intrinsic as well as extrinsic sources. In the recent years, cell-cell adhesion receptors have turned out as important regulators of cellular polarization. By interacting with conserved cell polarity proteins, they regulate the recruitment of polarity complexes to specific sites of cell-cell adhesion. By initiating intracellular signaling cascades at those sites, they trigger their specific subcellular activation. Not surprisingly, cell-cell adhesion receptors regulate diverse aspects of cell polarity, including apico-basal polarity in epithelial and endothelial cells, front-to-rear polarity in collectively migrating cells, and planar cell polarity during organ development. Here, we review the recent developments highlighting the central roles of cell-cell adhesion molecules in the development of cell polarity.

MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development.

Correct orientation of the mitotic spindle determines the plane of cellular cleavage and is crucial for organ development. In the developing cerebral cortex, spindle orientation defects result in severe neurodevelopmental disorders, but the precise mechanisms that control this important event are not fully understood. Here, we use a combination of high-content screening and mouse genetics to identify the miR-34/449 family as key regulators of mitotic spindle orientation in the developing cerebral cortex. By screening through all cortically expressed miRNAs in HeLa cells, we show that several members of the miR-34/449 family control mitotic duration and spindle rotation. Analysis of miR-34/449 knockout (KO) mouse embryos demonstrates significant spindle misorientation phenotypes in cortical progenitors, resulting in an excess of radial glia cells at the expense of intermediate progenitors and a significant delay in neurogenesis. We identify the junction adhesion molecule-A (JAM-A) as a key target for miR-34/449 in the developing cortex that might be responsible for those defects. Our data indicate that miRNA-dependent regulation of mitotic spindle orientation is crucial for cell fate specification during mammalian neurogenesis.

Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease.

Tetraspanins comprise a family of proteins embedded in the membrane through four transmembrane domains. One of the most distinctive features of tetraspanins is their ability to interact with other proteins in the membrane using their extracellular, transmembrane and cytoplasmic domains, allowing them to incorporate several proteins into clusters called tetraspanin-enriched microdomains. The spatial proximity of signaling proteins and their regulators enables a rapid functional cross-talk between these proteins, which is required for a rapid translation of extracellular signals into intracellular signaling cascades. In this article, we highlight a few examples that illustrate how tetraspanin-mediated interactions between cell surface proteins allow their functional cross-talk to regulate intracellular signaling.

Junctional adhesion molecule-A: functional diversity through molecular promiscuity.

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.

The regulation of junctional actin dynamics by cell adhesion receptors.

The formation of cell-cell junctions and the development of stable cell-cell adhesion require the association of actin filaments with the sites of cell-cell adhesion. From the initial formation of cell-cell junctions, which appear as punctate, spot-like junctions, to the formation of a stable actin belt that runs adjacent to cell-cell junctions, the actin cytoskeleton is closely associated with the adhesion apparatus. Importantly, the junctional actin is highly dynamic, even after the maturation of intercellular junctions and the development of apico-basal polarity. Regulators of both branched actin networks and of linear actin cables have been identified at cell-cell junctions, in particular at adherens junctions but also at tight junctions. These regulators of actin dynamics are often directly or indirectly associated with cell adhesion receptors, suggesting a critical role for cell adhesion molecules for the recruitment of regulators of actin dynamics to cell-cell junctions. Here, we review the recent developments on the role of cell adhesion molecules at epithelial and endothelial cell-cell junctions in the regulation of junctional actin dynamics.

Cell adhesion molecule control of planar spindle orientation.

Polarized epithelial cells align the mitotic spindle in the plane of the sheet to maintain tissue integrity and to prevent malignant transformation. The orientation of the spindle apparatus is regulated by the immobilization of the astral microtubules at the lateral cortex and depends on the precise localization of the dynein-dynactin motor protein complex which captures microtubule plus ends and generates pulling forces towards the centrosomes. Recent developments indicate that signals derived from intercellular junctions are required for the stable interaction of the dynein-dynactin complex with the cortex. Here, we review the molecular mechanisms that regulate planar spindle orientation in polarized epithelial cells and we illustrate how different cell adhesion molecules through distinct and non-overlapping mechanisms instruct the cells to align the mitotic spindle in the plane of the sheet.

Cell Adhesion at the Tight Junctions: New Aspects and New Functions.

Tight junctions (TJ) are cell-cell adhesive structures that define the permeability of barrier-forming epithelia and endothelia. In contrast to this seemingly static function, TJs display a surprisingly high molecular complexity and unexpected dynamic regulation, which allows the TJs to maintain a barrier in the presence of physiological forces and in response to perturbations. Cell-cell adhesion receptors play key roles during the dynamic regulation of TJs. They connect individual cells within cellular sheets and link sites of cell-cell contacts to the underlying actin cytoskeleton. Recent findings support the roles of adhesion receptors in transmitting mechanical forces and promoting phase separation. In this review, we discuss the newly discovered functions of cell adhesion receptors localized at the TJs and their role in the regulation of the barrier function.

A micropattern-based assay to study contact inhibition of locomotion and entosis of adherent human and canine cells in vitro.

We present a protocol for using micropatterns to study post-collision locomotion and entosis of human and canine cells in vitro. We describe steps for lentiviral transduction and the preparation of micropatterned slides consisting of narrow matrix-coated stripes separated by cytophobic spacers. We then detail cell seeding, chamber assembly, and live cell analysis. We provide steps for analysis by live cell imaging using fluorescence microscopy as well as fixing for subsequent analysis by confocal microscopy or correlative light and electron microscopy. For complete details on the use and execution of this protocol, please refer to Kummer et al. (2022)1 and Schwietzer et al. (2022).2.

TMIGD1: Emerging functions of a tumor supressor and adhesion receptor.

The development of multicellular organisms depends on cell adhesion molecules (CAMs) that connect cells to build tissues. The immunoglobulin superfamily (IgSF) constitutes one of the largest families of CAMs. Members of this family regulate such diverse processes like synapse formation, spermatogenesis, leukocyte-endothelial interactions, or epithelial cell-cell adhesion. Through their extracellular domains, they undergo homophilic and heterophilic interactions in cis and trans. Their cytoplasmic domains frequently bind scaffolding proteins to assemble signaling complexes. Transmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a IgSF member with two Ig-like domains and a short cytoplasmic tail that contains a PDZ domain-binding motif. Recent observations indicate that TMIGD1 has pleiotropic functions in epithelial cells and has a critical role in suppressing malignant cell behavior. Here, we review the molecular characteristics of TMIGD1, its interaction with cytoplasmic scaffolding proteins, the regulation of its expression, and its downregulation in colorectal and renal cancers.

Membrane recruitment of the polarity protein Scribble by the cell adhesion receptor TMIGD1.

Scribble (Scrib) is a multidomain polarity protein and member of the leucine-rich repeat and PDZ domain (LAP) protein family. A loss of Scrib expression is associated with disturbed apical-basal polarity and tumor formation. The tumor-suppressive activity of Scrib correlates with its membrane localization. Despite the identification of numerous Scrib-interacting proteins, the mechanisms regulating its membrane recruitment are not fully understood. Here, we identify the cell adhesion receptor TMIGD1 as a membrane anchor of Scrib. TMIGD1 directly interacts with Scrib through a PDZ domain-mediated interaction and recruits Scrib to the lateral membrane domain in epithelial cells. We characterize the association of TMIGD1 with each Scrib PDZ domain and describe the crystal structure of the TMIGD1 C-terminal peptide complexed with PDZ domain 1 of Scrib. Our findings describe a mechanism of Scrib membrane localization and contribute to the understanding of the tumor-suppressive activity of Scrib.

Rho and Rab Family Small GTPases in the Regulation of Membrane Polarity in Epithelial Cells.

Membrane polarity, defined as the asymmetric distribution of lipids and proteins in the plasma membrane, is a critical prerequisite for the development of multicellular tissues, such as epithelia and endothelia. Membrane polarity is regulated by polarized trafficking of membrane components to specific membrane domains and requires the presence of intramembrane diffusion barriers that prevent the intermixing of asymmetrically distributed membrane components. This intramembrane diffusion barrier is localized at the tight junctions (TJs) in these cells. Both the formation of cell-cell junctions and the polarized traffic of membrane proteins and lipids are regulated by Rho and Rab family small GTPases. In this review article, we will summarize the recent developments in the regulation of apico-basal membrane polarity by polarized membrane traffic and the formation of the intramembrane diffusion barrier in epithelial cells with a particular focus on the role of Rho and Rab family small GTPases.

Loss of contact inhibition of locomotion in the absence of JAM-A promotes entotic cell engulfment.

Entosis is a cell competition process during which tumor cells engulf other tumor cells. It is initiated by metabolic stress or by loss of matrix adhesion, and it provides the winning cell with resources derived from the internalized cell. Using micropatterns as substrates for single cell migration, we find that the depletion of the cell adhesion receptor JAM-A strongly increases the rate of entosis in matrix-adherent cells. The activity of JAM-A in suppressing entosis depends on phosphorylation at Tyr280, which is a binding site for C-terminal Src kinase, and which we have previously found to regulate tumor cell motility and contact inhibition of locomotion (CIL). Loss of JAM-A triggers entosis in matrix-adherent cells but not matrix-deprived cells. Our findings strongly suggest that the increased motility and the perturbed CIL response after the depletion of JAM-A promote entotic cell engulfment, and they link a dysregulation of CIL to entosis in breast cancer cells.

Intestinal brush border formation requires a TMIGD1-based intermicrovillar adhesion complex.

Intestinal epithelial cells absorb nutrients through the brush border, composed of dense arrays of highly ordered microvilli at their apical membranes. A protocadherin-based intermicrovillar adhesion complex localized at microvilli tips mediates microvilli packing and organization. Here, we identified a second adhesion complex localized at the proximal base region of microvilli. This complex contained the immunoglobulin superfamily member TMIGD1, which directly interacted with the microvillar scaffolding proteins EBP50 and E3KARP. Complex formation with EBP50 required the activation of EBP50 by the actin-binding protein ezrin and was enhanced by the dephosphorylation of Ser162 in the PDZ2 domain of EBP50 by the phosphatase PP1α. Binding of the EBP50-ezrin complex to TMIGD1 enhanced the dynamic turnover of EBP50 at microvilli. Enterocyte-specific inactivation of Tmigd1 in mice resulted in microvillar blebbing, loss of intermicrovillar adhesion, and perturbed brush border formation. Thus, we identified a second adhesion complex in microvilli and propose a mechanism that promotes microvillar formation and dynamics.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

JAM-A is a novel surface marker for NG2-Glia in the adult mouse brain.

BACKGROUND: Junctional adhesion molecule-A (JAM-A) is an adhesive protein expressed in various cell types. JAM-A localizes to the tight junctions between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesion and to the control of paracellular permeability. RESULTS: So far, the expression pattern of JAM-A has not been described in detail for the different cell types of the adult brain. Here we show that a subset of proliferating cells in the adult mouse brain express JAM-A. We further clarify that these cells belong to the lineage of NG2-glia cells. Although these mitotic NG2-glia cells express JAM-A, the protein never shows a polarized subcellular distribution. Also non-mitotic NG2-glia cells express JAM-A in a non-polarized pattern on their surface. CONCLUSIONS: Our data show that JAM-A is a novel surface marker for NG2-glia cells of the adult brain.

Spermatid differentiation requires the assembly of a cell polarity complex downstream of junctional adhesion molecule-C.

During spermatogenesis in the mammalian testis, stem cells (spermatogonia) differentiate into spermatocytes, which subsequently undergo two consecutive meiotic divisions to give rise to haploid spermatids. These cells are initially round but progressively elongate, condense their nuclei, acquire flagellar and acrosomal structures, and shed a significant amount of their cytoplasm to form spermatozoa (the sperm cells) in a developmental cascade termed spermiogenesis. Defects in these processes will lead to a lack of mature sperm cells (azoospermia), which is a major cause of male infertility in the human population. Here we report that a cell-surface protein of the immunoglobulin superfamily, junctional adhesion molecule-C (JAM-C), is critically required for the differentiation of round spermatids into spermatozoa in mice. We found that Jam-C is essential for the polarization of round spermatids, a function that we attribute to its role in the assembly of a cell polarity complex.