From the Cover: Protein O-mannosylation is crucial for E-cadherin–mediated cell adhesion

In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell–cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell–cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation–deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell–cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl–conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.Protein O-mannosylation is a vital protein modification that is evolutionarily conserved across eukaryotes (1). In humans, defects in this modification result in a heterogeneous group of congenital muscular dystrophies (CMDs, α-dystroglycanopathies). The most severe of these disorders, Walker–Warburg syndrome, is characterized by CMD associated with brain malformations of various degrees, ocular abnormalities, and, most often, fatal outcome during the first year of life (2). In contrast, milder disorders such as limb-girdle muscular dystrophy, in which neither the brain nor the eyes are affected, may not present until adulthood (2). The key pathological feature of these diseases is the lack of O-mannosyl glycans on α-dystroglycan (α-DG), an integral component of the dystrophin–glycoprotein complex (2, 3). In the absence of these glycans, binding of α-DG to its extracellular matrix ligands (e.g., laminin) is abolished, and, consequently, basement membranes are fragmented (3–5). In addition to α-DG, O-mannosyl glycans constitute up to 30% of total O-linked carbohydrates in the mammalian brain (6, 7). However, to date only a few other proteins [including CD24 (8), PTPRZ1 (9), neurofascin 186 (10), neurocan, and versican (11)] have been shown to undergo O-mannosylation.Synthesis of O-mannosyl glycans is initiated in the endoplasmic reticulum by the transfer of mannose from dolichol monophosphate-activated mannose (Dol-P-Man) to serine or threonine residues on membrane and secretory proteins (1). In mammals, this reaction is catalyzed by a heteromeric complex of the protein O-mannosyltransferase 1 (POMT1) and 2 (POMT2) (12). We previously showed that Pomt1-null mice display embryonic lethality during postimplantation development, between embryonic day (E)7.5 and E9.5 because of abnormal glycosylation and maturation of α-DG (4). Similarly, knockdown of POMTs in zebrafish and Drosophila melanogaster leads to severe developmental defects that are largely attributable to dysfunctional α-DG (13, 14). Although these animal models have been very helpful in elucidating the role of α-DG–linked O-mannosyl glycans, they have not revealed other biological functions of this protein modification.In both animals and humans, adhesive interactions between neighboring cells are essential for embryogenesis as well as for tissue morphogenesis and renewal (15). Adherens junctions are sites of cell–cell contact where cell-surface receptors of the cadherin family mediate adhesion (16). Classical cadherins are conserved among vertebrates and invertebrates (17). These plasma-membrane glycoproteins share a conserved cytoplasmic domain, a single-pass transmembrane domain, and an ectodomain containing five extracellular cadherin (EC) domains (18). Located on opposing cells, cadherin ectodomains form calcium-dependent homophilic interactions whereby they mediate cell–cell contact (18). The classical cadherin family comprises multiple members, including epithelial cadherin (E-cad, CDH1), neuronal cadherin (N-cad, CDH2), and retinal cadherin (R-cad, CDH4), each of which shows a distinct tissue-specific distribution pattern (16). E-cad plays a critical role in the epithelial–mesenchymal transition during embryogenesis; E-cad–knockout mice die before implantation because of the lack of a functional trophectodermal cell layer at the blastocyst stage (19). Cell–cell adhesion is crucial not only for development but also for tissue morphogenesis and for the invasiveness of human cancer cells (20). Thus, it is particularly important to assess, on a molecular level, the factors that affect this process.In the present study we demonstrate that protein O-mannosylation is essential for the formation of adherens junctions in the preimplantation embryo. Embryos deficient for O-mannosylation die during the morula-to-blastocyst transition because of impaired blastomere adhesion. We identify E-cad as an O-mannosylated glycoprotein and show that this modification is essential for cadherin-mediated cell adhesion. Our identification of functionally relevant O-mannosyl glycans on cadherins is expected to provide further insights into the molecular pathologies of α-dystroglycanopathies.     

Inhibition of the K+ channel KCa3.1 ameliorates T cell–mediated colitis

The calcium-activated K⁺ channel KCa3.1 plays an important role in T lymphocyte Ca²⁺ signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca²⁺ influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1^−/− mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1^−/− mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor–stimulated Ca²⁺ influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca²⁺ influx and cytokine production were impaired in KCa3.1^−/− Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1^−/− protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naïve CD4 T cells into rag2^−/− recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis.     

Controlling cell–cell interactions using surface acoustic waves

The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.Multicellular systems rely on the interaction between cells to coordinate cell signaling and regulate cell functions. Understanding the mechanism and process of cell–cell interaction is critical to many physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes (1–3). Despite significant advances in this field, to further understand how cells interact and communicate with each other, a robust, biocompatible method to precisely control the spatial and temporal association of cells and to create defined cellular assemblies is urgently needed (4). Although several methods have been used to pattern cells, limitations still exist for the demonstrated methods including those that make use of optical, electrical, magnetic, hydrodynamic, and contact printing technologies (5–9). Firstly, most of the methods require modification of the cell’s native state. The magnetic assembly method, for example, requires cells to be labeled with magnetic probes. Dielectrophoresis typically requires the use of a special medium (e.g., nonconductive) which may lack essential nutrients or have biophysical properties (such as the osmolality) that may adversely affect cell growth or physiology (6). Optical tweezers provide a label-free and contactless approach, but typically require high laser power to manipulate cells, leading to a high risk of cell damage (5). Secondly, the working principles of the existing technologies mostly preclude the combination of high precision and high throughput into a single device. It is difficult for high-throughput methods (such as magnetic assemblies) to achieve single-cell level precision, whereas the high-precision methods often require complex experimental setup to manipulate multiple cells simultaneously. Thirdly, most of the existing methodologies lack the ability to maintain cell assemblies in suspension, thereby limiting the application of these methods for the study of cell–cell and cell–matrix interactions.As an alternative to using optical, electrical, or magnetic forces to manipulate cells, it has been demonstrated that biological specimens can also be manipulated using acoustic forces (10–16). Acoustic force can be applied through either bulk acoustic waves (BAWs) or surface acoustic waves (SAWs). Compared with the conventional BAW-based approaches (12, 13), SAW-based approaches (14–16) are becoming increasingly important in applications in cell biology and medicine as SAWs allow simpler device fabrication and experimental setup, higher manipulation resolution and flexibility, and better compatibility with optical imaging systems (allowing use of transparent devices). Thus far, the SAW-based approach has been reported to be able to manipulate single cells (15), but it has not yet been demonstrated for controlling cell–cell distance and interactions. This is mainly due to the difficulties in achieving a sufficient level of regulation of pressure nodes, which is needed to control the position of the cells with a high degree of precision. In this study, we demonstrate a SAW device that can accurately and reproducibly control pressure nodes and perform various functions for cell–cell interaction studies. Through superposing two orthogonal standing SAWs with differential input frequencies, we achieved highly regulated dot-array configuration of pressure nodes that facilitate high-precision control of cell–cell interactions, rather than the net-array pressure node configurations used in previous SAW devices (14, 15). This acoustic tweezers cell-manipulation method does not require any modification of the growth conditions, allowing cells to be cultured in their native media. It is highly adaptable to the requirements of various applications and is capable of delivering both high precision (controlling intercellular distance at the micrometer scale) and high throughput (forming thousands of cell assemblies with tunable geometric configurations) in a single device. In addition, our method offers unprecedented flexibility over the control of cell assemblies. The geometry of cell assemblies can be finely tuned by changing the acoustic field. Moreover, the system is capable of holding cell assemblies in suspension at precise locations while assessing their biological functions without the use of permanent structures. These suspended cell assemblies can be allowed to settle to the surface to adhere and disperse. To demonstrate the power of this technology, we applied the system to explore gap junctional intercellular communication (GJIC) and quantitatively investigated various forms of functional intercellular communication by visualizing gap junctional dye exchange among coupled cells.     

Matrix cross-linking–mediated mechanotransduction promotes posttraumatic osteoarthritis

Osteoarthritis (OA) is characterized by impairment of the load-bearing function of articular cartilage. OA cartilage matrix undergoes extensive biophysical remodeling characterized by decreased compliance. In this study, we elucidate the mechanistic origin of matrix remodeling and the downstream mechanotransduction pathway and further demonstrate an active role of this mechanism in OA pathogenesis. Aging and mechanical stress, the two major risk factors of OA, promote cartilage matrix stiffening through the accumulation of advanced glycation end-products and up-regulation of the collagen cross-linking enzyme lysyl oxidase, respectively. Increasing matrix stiffness substantially disrupts the homeostatic balance between chondrocyte catabolism and anabolism via the Rho–Rho kinase–myosin light chain axis, consequently eliciting OA pathogenesis in mice. Experimental enhancement of nonenzymatic or enzymatic matrix cross-linking augments surgically induced OA pathogenesis in mice, and suppressing these events effectively inhibits OA with concomitant modulation of matrix degrading enzymes. Based on these findings, we propose a central role of matrix-mediated mechanotransduction in OA pathogenesis.The mechanics of the ECM and resulting effects on its interactions with cells regulate numerous biological functions (1). Various pathological conditions in human diseases are associated with aberrant ECM remodeling and consequent deviation from intrinsic ECM material properties (2). Mechanical perturbation of ECM affects the ways in which cells respond to externally applied mechanical forces and generate internal traction forces through cell–matrix interactions (3). Therefore, elucidation of the functional relationships between ECM mechanics and cellular transduction pathways is of critical importance.Articular cartilage ECM consisting of a collagenous network and highly charged proteoglycans confers the unique load-bearing function to joints. The dense aggregates of negatively charged proteoglycans provide resistance to compressive loading by promoting osmotic swelling, which is counterbalanced by cross-linked collagen fibrils that confer tissue tensile strength. Disruption of this delicate balance leads to structural damage and functional failure of articular cartilage and, consequently, to development of osteoarthritis (OA), the most common arthropathy (4, 5). OA cartilage ECM undergoes extensive remodeling, characterized by a decrease in matrix compliance (6, 7). These changes occur at the level of individual collagen fibrils, although the precise mechanisms regulating matrix remodeling remain elusive. Notably, matrix remodeling precedes cartilage destruction (6, 7), suggesting that monitoring the mechanical properties of cartilage matrix could serve as an innovative diagnostic approach for early detection of OA. Significant influence of matrix stiffness on mesenchymal lineage specification has been documented, and data have been obtained on the optimal ranges of substrate rigidity promoting osteogenesis. This regulatory process requires nonmuscle myosin II activity, with concomitant effects on adhesion and actin cytoskeleton structures (8).In this study, we sought to determine molecular mechanisms leading to ECM remodeling over the course of OA development and to investigate how mechanical alterations in cartilage matrix affect chondrocyte metabolism and regulate OA pathogenesis.     

B7-H1 shapes T-cell–mediated brain endothelial cell dysfunction and regional encephalitogenicity in spontaneous CNS autoimmunity

Molecular mechanisms that determine lesion localization or phenotype variation in multiple sclerosis are mostly unidentified. Although transmigration of activated encephalitogenic T cells across the blood–brain barrier (BBB) is a crucial step in the disease pathogenesis of CNS autoimmunity, the consequences on brain endothelial barrier integrity upon interaction with such T cells and subsequent lesion formation and distribution are largely unknown. We made use of a transgenic spontaneous mouse model of CNS autoimmunity characterized by inflammatory demyelinating lesions confined to optic nerves and spinal cord (OSE mice). Genetic ablation of a single immune-regulatory molecule in this model [i.e., B7-homolog 1 (B7-H1, PD-L1)] not only significantly increased incidence of spontaneous CNS autoimmunity and aggravated disease course, especially in the later stages of disease, but also importantly resulted in encephalitogenic T-cell infiltration and lesion formation in normally unaffected brain regions, such as the cerebrum and cerebellum. Interestingly, B7-H1 ablation on myelin oligodendrocyte glycoprotein-specific CD4⁺ T cells, but not on antigen-presenting cells, amplified T-cell effector functions, such as IFN-γ and granzyme B production. Therefore, these T cells were rendered more capable of eliciting cell contact-dependent brain endothelial cell dysfunction and increased barrier permeability in an in vitro model of the BBB. Our findings suggest that a single immune-regulatory molecule on T cells can be ultimately responsible for localized BBB breakdown, and thus substantial changes in lesion topography in the context of CNS autoimmunity.Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the CNS. Disease pathogenesis is initiated by peripheral activation of autoimmune T lymphocytes by yet unknown mechanisms, followed by T-cell expansion and subsequent migration across the complex structure of the blood–brain barrier (BBB). Within the CNS, entry of this first wave of T cells elicits recruitment of other immune cells, which together evoke a local inflammatory process ultimately resulting in demyelination, as well as axonal and neuronal damage (1). Several histological MS subtypes have been described with regard to lesion distribution and cellular composition (2). The reasons underlying distinct lesion development at different anatomical sites still remain however largely elusive: Some authors have proposed that the nature and expression pattern of the target autoantigen might play a role (3, 4). Others have observed an influence of the HLA complex and its role in shaping antigen presentation, thus suggesting that T-cell antigen specificity might impact the location of inflammation (5). Additionally, T-cell polarization into distinct T helper subtypes, as well as their expression pattern of chemokine receptors and adhesion molecules, has been implicated in determining the localization of inflammatory lesions within the CNS (6–8).With respect to the very onset of lesion development, imaging techniques have revealed that a local dysregulation of the BBB integrity already precedes lesion formation (9, 10). Moreover, in a recent study, Maggi et al. observed an association between early changes in BBB permeability and perivascular inflammatory cuffing (11). However, the mechanisms causing local BBB dysfunction as an initial step in MS lesion formation still remain to be resolved.Animal models of CNS autoimmunity, especially the models of experimental autoimmune encephalomyelitis (EAE), have been of great value in elucidating crucial steps in disease pathogenesis. Up to now, a broad range of different models with distinct features have been available, the most commonly used of them induced by immunization of susceptible rodents with myelin antigens or adoptive transfer of highly activated myelin-reactive T cells. However, these models are of limited value for studying the very first steps of disease initiation and lesion development, mainly due to their dependence on microbial adjuvants for artificial breakdown of tolerance and promotion of BBB disruption (12–14). During the last years, novel models of spontaneous CNS autoimmunity have been developed by crossing myelin-specific T-cell receptor (TCR) transgenic mice and myelin-specific Ig heavy chain knock-in mice (3, 15). The offspring of those mice are characterized by spontaneous development of a severe form of EAE. Lesions here are confined to the spinal cord and optic nerves, making this model an interesting tool for further investigation of the determinants of lesion development and lesion distribution under “homeostatic”, non–vaccine-depending immune-regulatory conditions.We investigated the hypothesis whether and how genetic modification of single immune-regulatory molecules could influence lesion distribution and phenotype variation in genetically susceptible hosts. We thus used the opticospinal EAE (OSE) model and genetically modulated an immune-regulatory molecule, B7-homolog 1 (B7-H1). Absence of B7-H1 increased incidence of spontaneous CNS autoimmunity and aggravated disease course. Importantly, B7-H1 ablation on T cells, but not on antigen-presenting cells (APCs), enhanced their capacity to elicit brain endothelial cell (EC) dysfunction in a cell contact-dependent fashion in an in vitro model of the BBB and increased BBB permeability both in vitro and in vivo. Our findings suggest that single immune regulatory molecules that alter the activation status of encephalitogenic T cells can influence lesion distribution, which is associated with an altered capacity of these cells to elicit transient focal EC dysfunction and barrier dysfunction as essential steps for lesion development.     

Enhanced receptor–clathrin interactions induced by N-glycan–mediated membrane micropatterning

Glycan–protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN–clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.Glycans are fundamental cellular components ubiquitously present in the extracellular matrix and cell membrane as glycoproteins or glycolipids. Glycan-binding proteins such as galectins, siglecs, and selectins are mostly multivalent and thus thought to cross-link glycoproteins into higher-order aggregates, creating a cell surface glycan-based connectivity also called glycan lattice or network (1–3). By concentrating specific glycoproteins or glycolipids while excluding other cell surface molecules, surface glycan-based connectivity can organize the plasma membrane into specialized domains that perform unique functions (1, 3–6). Nevertheless, direct observation of glycan-mediated ligand cross-linking in living cells remains challenging (7). Notwithstanding, there is no doubt that surface glycan-based connectivity is essential in the control of multiple biological processes including immune cell activation and homeostasis, cell proliferation and differentiation, and receptor turnover and endocytosis (1, 5, 6, 8).Clathrin-mediated endocytosis (CME) constitutes the primary pathway of cargo internalization in mammalian cells regulating the surface expression of receptors (9). Formation of clathrin-coated pits (CCPs) starts by nucleation of coat assembly at distributed positions in the inner surface of the plasma membrane, where it continues to grow or dissolve rapidly unless coat stabilization occurs (10, 11). One event that clearly correlates with successful CCP stabilization is cargo loading (11). Recent studies show that cargo molecules diffuse randomly on the cell membrane until they meet growing CCPs, with the extent of cargo interactions regulating CCP maturation (12). As such, factors that affect cargo mobility within/at the cell surface will inevitably impact on CCP maturation and successful internalization. In the context of surface glycan–protein interactions, it has been shown that glycoproteins with an intact glycan-based connectivity exhibit reduced lateral mobility and this correlates with compromised endocytosis (3, 13–17). How this precisely occurs is poorly defined, although fluorescence recovery after photobleaching on the EGF receptor (EGFR) suggested that cell surface glycan-based interactions restrict EGFR dynamics and localization into membrane regions away from endocytic platforms (14, 17). Whether this is a general mechanism for glycosylated proteins or specific to EGFR is not known. Moreover, visualization of receptor interactions with the endocytic machinery under the influence of the glycan network has not yet been attained.In this work we applied superresolution nanoscopy and developed a dedicated dual-color single-molecule spatio-dynamic exploration approach to visualize the impact of glycan-based interactions on the spatiotemporal organization and clathrin interaction of a glycosylated membrane receptor involved in pathogen recognition and uptake. We focused on the transmembrane glycoprotein dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) given its importance in supporting primary immune responses such as pathogen recognition and uptake on immature dendritic cells (imDCs), signaling, and cell adhesion (6, 18–20). Moreover, DC-SIGN contains a single N-glycosylation site, organizes in nanoclusters at the cell membrane (19, 21–23), and internalizes bound antigens via CPPs for subsequent processing and presentation to T cells (20, 24–26). Our work provides insights on how surface glycan-mediated interactions tune spatiotemporal micropatterning of receptors on the cell membrane, potentially regulating interactions with the endocytic machinery and underscoring the importance and complex role of surface glycans on cell membrane organization and function.     

Impaired autophagy leads to abnormal dendritic cell–epithelial cell interactions

Background and aimsDendritic cells (DC) are key players in intestinal immunity, as these cells can direct the immune response to either a tolerogenic or an immunogenic phenotype. In the intestine, DC sample and process luminal antigens by protruding dendrites through the epithelial cell layer. At the same time barrier integrity is maintained through the continuous formation of tight junctions. Aberrations in these interactions may lead to altered antigen sampling and improper immune responses. We have recently shown that autophagy, a process implicated in the pathogenesis of Crohn's disease, regulates cellular interactions in the context of DC and T cells. In this study we aimed to determine whether autophagy also regulates DC–epithelial cell interactions and whether this influences the ensuing immune response.MethodsDC were generated from peripheral blood monocytes of healthy volunteers. For interaction studies, DC were co-cultured with intestinal epithelial cells on the baso-lateral side of a transwell insert. Modulation of autophagy was achieved using atg16l1 specific siRNA or pharmacological inhibitors. Intraepithelial protrusion of dendrites was determined by confocal microscopy. Luminal sampling and DC activation status were analyzed by flow cytometry. Protein expression was measured by immunoblotting and cytometric bead assay.ResultsAdhesion molecules E-cadherin and occludin partly localized to autophagosomes and increased autophagy resulted in decreased levels of these proteins. Reduced autophagy in either DC, epithelial cells or both resulted in the decreased formation of transepithelial protrusions by DC as well as a reduction in antigen sampling. Moreover, when autophagy was inhibited in the co-culture model, DC expressed increased levels of HLA-DR and costimulatory molecule CD86. Furthermore, decreased levels of autophagy resulted in lower IL-10 production by DC and these cells induced significantly more T-cell proliferation in an allogeneic mixed lymphocyte reaction.ConclusionsIn intestinal DC–epithelial cell interactions, autophagy deficiency leads to decreased antigen sampling, increased DC maturation and a more pro-inflammatory type of DC.     

Mechanical tugging force regulates the size of cell–cell junctions

Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell–matrix and cell–cell contact. While increased mechanical loading at cell–matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell–cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell–cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell–cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell–cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.     

A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.Proline-rich segments (PRSs) belong to the most abundant sequence motifs of the proteome (1), interacting frequently with PRS-recognizing domains (PRDs), such as EVH1, SH3, GYF, and WW. Although exhibiting different tertiary structures, PRDs expose clusters of aromatic residues, forming a shallow, corrugated binding groove with a hydrogen bond-donating residue (W, Y) in the central position. In the bound state, PRSs often show a conformation closely related to the ideal left-handed polyproline II (PPII) helix characterized by backbone angles of Φ = −78° and Ψ = +146° (2). As a consequence of the axial symmetry of PPII helices, two different types of consensus motifs occur: one containing PxxP specifically recognized by the EVH1 and SH3 domains, the other comprising xPPx, typical for motifs binding at WW and GYF domains. The conserved prolines represent the core of the consensus motifs and interact intimately with the exposed aromatic side chains. They cannot be replaced by any other natural amino acid without complete loss of affinity (2, 3). On the other hand, the core motif alone binds only very weakly to its PRD. Further interactions of flanking residues located outside the core motif contribute substantially to both affinity and specificity. Incorporation of nonnatural amino acids in place of such specificity-determining residues is therefore often beneficial for binding (4–9). However, peptide ligands display a number of disadvantages when used as competitors, among them metabolic instability and often low cell permeability. Cell-permeable small molecules that grant the ability to modulate the function of PRDs are still not available.Here, we present a modular concept for the systematic development of such low-molecular weight compounds. It is based on molecular building blocks that can replace the conserved prolines within the core motif without any loss of affinity. Combinations of such building blocks allow complete replacement of the proline-rich core motifs. They may be supplemented with organic scaffolds addressing the flanking epitopes to obtain peptidomimetic inhibitors of PRDs, highly desirable for functional analysis of PRS-mediated protein–protein interactions.As proof of concept, we developed a peptidomimetic inhibitor targeting the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family Ena/VASP homology 1 (EVH1) domains. This protein family is involved in modulation of the actin cytoskeleton, a complex and highly regulated process, which is the driving force of directed cell migration (10, 11) and plays important roles in disease-relevant processes like tumor metastasis (12, 13). The Ena/VASP family proteins [i.e., VASP, enabled homolog (EnaH), and Ena-VASP–like (EVL) (14–16)] are notably localized at focal adhesions and lamellipodia. Single Ena/VASP protein deletions are mostly compensated for the other members of the family (17); however, triple knock-out mice are embryonic lethal (18, 19). The proteins comprise EVH1 and Ena/VASP homology 2 (EVH2) domains, separated by a proline-rich region. Although EVH2 binds to the barbed ends of actin filaments, EVH1 interacts with proteins, like zyxin or lamellipodin (Lpd also called RAPH1), that contain the class 1 EVH1 consensus motif [FYWL]P.ϕP (ϕ is an aliphatic amino acid) (2, 20–22). Using our peptidomimetic inhibitor, we show that inhibition of the Ena/VASP family EVH1 domains strongly influences both cellular localization of VASP as well as cell migration.     

c-Ret–mediated hearing loss in mice with Hirschsprung disease

A significantly increased risk for dominant sensorineural deafness in patients who have Hirschsprung disease (HSCR) caused by endothelin receptor type B and SOX10 has been reported. Despite the fact that c-RET is the most frequent causal gene of HSCR, it has not been determined whether impairments of c-Ret and c-RET cause congenital deafness in mice and humans. Here, we show that impaired phosphorylation of c-Ret at tyrosine 1062 causes HSCR-linked syndromic congenital deafness in c-Ret knockin (KI) mice. The deafness involves neurodegeneration of spiral ganglion neurons (SGNs) with not only impaired phosphorylation of Akt and NF-κB but decreased expression of calbindin D28k in inner ears. The congenital deafness involving neurodegeneration of SGNs in c-Ret KI mice was rescued by introducing constitutively activated RET. Taken together with our results for three patients with congenital deafness with c-RET–mediated severe HSCR, our results indicate that c-Ret and c-RET are a deafness-related molecule in mice and humans.     

Rosuvastatin inhibits the smooth muscle cell proliferation by targeting TNFα mediated Rho kinase pathway

Objective To investigate whether Tumor Necrosis Factor-alpha (TNFα) is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and clarify the mechanism and intervention manner of anti-atherosclerosis by Rosuvastatin. Methods Wistar neonate rat smooth muscle cells were cultured, and the activity of cell proliferation was determined by methyl thiazolyl tetrazolium (MTT). The expression of Rho kinase genes after the stimulation of TNFα was evaluated by RT-PCR. Western blot method was used to measure the protein expression of proliferating cell nuclear antigen (PCNA) after TNFα stimulation and Rosuvastatin intervention in smooth muscle cell. Results The TNFα stimulation significantly enhanced the expression of Rho kinase and increased the expression of PCNA protein in smooth muscle cells (P < 0.05). These effects were positively correlated with prolonged treatment whereas additional Rosuvastatin administration inhibited the above-mentioned effects (P < 0.05). Conclusions The activation of TNFα mediated Rho kinase signaling pathway can significantly promote smooth muscle cell proliferation, and Rosuvastatin can not only inhibit this pathway but also the induced proliferation.