Extracellular leucine-rich repeat proteins are required to organize the apical extracellular matrix and maintain epithelial junction integrity in C. elegans

Epithelial cells are linked by apicolateral junctions that are essential for tissue integrity. Epithelial cells also secrete a specialized apical extracellular matrix (ECM) that serves as a protective barrier. Some components of the apical ECM, such as mucins, can influence epithelial junction remodeling and disassembly during epithelial-to-mesenchymal transition (EMT). However, the molecular composition and biological roles of the apical ECM are not well understood. We identified a set of extracellular leucine-rich repeat only (eLRRon) proteins in C. elegans (LET-4 and EGG-6) that are expressed on the apical surfaces of epidermal cells and some tubular epithelia, including the excretory duct and pore. A previously characterized paralog, SYM-1, is also expressed in epidermal cells and secreted into the apical ECM. Related mammalian eLRRon proteins, such as decorin or LRRTM1-3, influence stromal ECM or synaptic junction organization, respectively. Mutants lacking one or more of the C. elegans epithelial eLRRon proteins show multiple defects in apical ECM organization, consistent with these proteins contributing to the embryonic sheath and cuticular ECM. Furthermore, epithelial junctions initially form in the correct locations, but then rupture at the time of cuticle secretion and remodeling of cell-matrix interactions. This work identifies epithelial eLRRon proteins as important components and organizers of the pre-cuticular and cuticular apical ECM, and adds to the small but growing body of evidence linking the apical ECM to epithelial junction stability. We propose that eLRRon-dependent apical ECM organization contributes to cell-cell adhesion and may modulate epithelial junction dynamics in both normal and disease situations.     

Tight junctions as targets of infectious agents

The epithelial barrier is a critical border that segregates luminal material from entering tissues. Essential components of this epithelial fence are physical intercellular structures termed tight junctions. These junctions use a variety of transmembrane proteins coupled with cytoplasmic adaptors, and the actin cytoskeleton, to attach adjacent cells together thereby forming intercellular seals. Breaching of this barrier has profound effects on human health and disease, as barrier deficiencies have been linked with the onset of inflammation, diarrhea generation and pathogenic effects. Although tight junctions efficiently restrict most microbes from penetrating into deeper tissues and contain the microbiota, some pathogens have developed specific strategies to alter or disrupt these structures as part of their pathogenesis, resulting in either pathogen penetration, or other consequences such as diarrhea. Understanding the strategies that microorganisms use to commandeer the functions of tight junctions is an active area of research in microbial pathogenesis. In this review we highlight and overview the tactics bacteria and viruses use to alter tight junctions during disease. Additionally, these studies have identified novel tight junction protein functions by using pathogens and their virulence factors as tools to study the cell biology of junctional structures.     

Adherens junctions: new insight into assembly,modulation and function

Adherens junctions play pivotal roles in cell and tissue organization and patterning by mediating cell adhesion and cell signaling. These junctions consist of large multiprotein complexes that join the actin cytoskeleton to the plasma membrane to form adhesive contacts between cells or between cells and extracellular matrix. The best-known adherens junction is the zonula adherens (ZA) that forms a belt surrounding the apical pole of epithelial cells. Recent studies in Drosophila have further illuminated the structure of adherens junctions. Scaffolding proteins encoded by the stardust gene are novel components of the Crumbs complex, which plays a critical role in ZA assembly.1-3 The small GTPase Rap1 controls the symmetric re-assembly of the ZA after cell division.4 Finally, the asymmetric distribution of adherens junction material regulates spindle orientation during asymmetric cell division in the sensory organ lineage.     

Desmin at myotendinous junctions

Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.     

Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies

We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, connexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termii of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on approximately 50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.     

Some observations on sperm transport through the uterotubal junction of the rat

Entry of spermatozoa into the oviducts of mammals is restricted by the uterotubal junctions. The extent to which these junctions act as selective valves, or filters, for sperm transport has not been determined. A new technique has been developed that permits the direct visualization of sperm transport through the uterotubal junction of the rat in vitro. After mating or artificial insemination, the female tract is removed to a special "observation dish" containing oxygenated Earle's solution maintained at 37 degrees C. The oviducts are severed 1.0 - 1.5 mm above the uterotubal junctions. Under appropriate magnification and with oblique transillumination, spermatozoa may be observed emerging from the cut ends. It was noted that only motile spermatozoa emerged and that they usually appeared individually, with an interval of several minutes between each. Their egress was not directly related to contractions of the uterine cornu. Neither immotile spermatozoa nor a dye solution were observed to pass through the uterotubal junction. It is concluded that sperm motility is important, and probably essential, for sperm entry into the oviducts in the rat. Scanning electron microscopy revealed that the rat uterotubal junction forms a small mound or papilla projecting into the uterine cavity. No ciliated cells were observed in this region.     

Continuous endocytic recycling of tight junction proteins: how and why?

Tight junctions consist of many proteins, including transmembrane and associated cytoplasmic proteins, which act to provide a barrier regulating transport across epithelial and endothelial tissues. These junctions are dynamic structures that are able to maintain barrier function during tissue remodelling and rapidly alter it in response to extracellular signals. Individual components of tight junctions also show dynamic behaviour, including migration within the junction and exchange in and out of the junctions. In addition, it is becoming clear that some tight junction proteins undergo continuous endocytosis and recycling back to the plasma membrane. Regulation of endocytic trafficking of junctional proteins may provide a way of rapidly remodelling junctions and will be the focus of this chapter.     

Morphology and distribution of cystic cavities in the normal murine thymus

Summary The normal murine thymus was examined by lightand electron microscopy to determine the distribution and morphology of extracellular cystic cavities. Most cavities were confined to the cranial half of each gland, situated at the junction between cortex and medulla. They varied in size and shape, and gave rise to narrow channels that coursed to the capsular surface of the gland. Large cavities could be divided into three zones. A short cranial zone exhibited gland-like features, consisting of cells lining a clear lumen. A central zone was lined by a diverse population of cells. Some possessed secretory granules, while others exhibited an apical ciliated border. Lining cells interdigitated with each other and were joined laterally by intercellular junctions. The lumen of the central zone contained lymphocytes and macrophages in an amorphous extracellular matrix. The caudal zone of each cavity had an attenuated and incomplete cellular lining, communicating directly with the surrounding thymic parenchyma. Thymic cavities may represent the initial part of the efferent lymphatic system of the gland, beginning in the tissue spaces at the corticomedullary junction. Selected cells could then enter and interact with the luminal contents in the central zone of the cavity. Ciliated cells may then propel lymphocytes and secretions into the narrow channels radiating from the uppermost part of the chamber, leaving a cell-free lumen in this region. These cavities may function in sequestering lymphocytes, macrophages and thymic secretions before their exit from the gland.     

The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

The herpes simplex virus (HSV) glycoprotein complex gE-gI mediates the spread of viruses between adjacent cells, and this property is especially evident for cells that form extensive cell junctions, e.g., epithelial cells, fibroblasts, and neurons. Mutants lacking gE or gI are not compromised in their ability to enter cells as extracellular viruses. Therefore, gE-gI functions specifically in the movement of virus across cell-cell contacts and, as such, provides a molecular handle on this poorly understood process. We expressed gE-gI in human epithelial cells by using replication-defective adenovirus (Ad) vectors. gE-gI accumulated at lateral surfaces of the epithelial cells, colocalizing with the adherens junction protein β-catenin but was not found on either the apical or basal plasma membranes and did not colocalize with ZO-1, a component of tight junctions. In subconfluent monolayers, gE-gI was found at cell junctions but was absent from those lateral surfaces not in contact with another cell, as was the case for β-catenin. Similar localization of gE-gI to cell junctions was observed in HSV-infected epithelial cells. By contrast, HSV glycoprotein gD, expressed using a recombinant Ad vectors, was found primarily along the apical surfaces of cells, with little or no protein found on the basal or lateral surfaces. Expression of gE-gI without other HSV polypeptides did not cause redistribution of either ZO-1 or β-catenin or alter tight-junction functions. Together these results support a model in which gE-gI accumulates at sites of cell-cell contact by interacting with junctional components. We hypothesize that gE-gI mediates transfer of HSV across cell junctions by virtue of these interactions with cell junction components.     

Under Construction: Building the Macromolecular Superstructure and Signaling Components of an Electrical Synapse

A great deal is now known about the protein components of tight junctions and adherens junctions, as well as how these are assembled. Less is known about the molecular framework of gap junctions, but these also have membrane specializations and are subject to regulation of their assembly and turnover. Thus, it is reasonable to consider that these three types of junctions may share macromolecular commonalities. Indeed, the tight junction scaffolding protein zonula occluden-1 (ZO-1) is also present at adherens and gap junctions, including neuronal gap junctions. On the basis of these earlier observations, we more recently found that two additional proteins, AF6 and MUPP1, known to be associated with ZO-1 at tight and adherens junctions, are also components of neuronal gap junctions in rodent brain and directly interact with connexin36 (Cx36) that forms these junctions. Here, we show by immunofluorescence labeling that the cytoskeletal-associated protein cingulin, commonly found at tight junctions, is also localized at neuronal gap junctions throughout the central nervous system. In consideration of known functions related to ZO-1, AF6, MUPP1, and cingulin, our results provide a context in which to examine functional relationships between these proteins at Cx36-containing electrical synapses in brain--specifically, how they may contribute to regulation of transmission at these synapses, and how they may govern gap junction channel assembly and/or disassembly.     

COEXISTENCE OF GAP AND SEPTATE JUNCTIONS IN AN INVERTEBRATE EPITHELIUM

The intercellular junctions of the epithelium lining the hepatic caecum of Daphnia were examined. Electron microscope investigations involved both conventionally fixed material and tissue exposed to a lanthanum tracer of the extracellular space. Both septate junctions and gap junctions occur between the cells studied. The septate junctions lie apically and resemble those commonly discerned between cells of other invertebrates. They are atypical in that the high electron opacity of the extracellular space obscures septa in routine preparations. The gap junctions are characterized by a uniform 30 A space between apposed cell membranes. Lanthanum treatment of gap junctions reveals an array of particles of 95 A diameter and 120 A separation lying in the plane of the junction. As this pattern closely resembles that described previously in vertebrates, it appears that the gap junction is phylogenetically widespread. In view of evidence that the gap junction mediates intercellular electrotonic coupling, the assignment of a coupling role to other junctions, notably the septate junction, must be questioned wherever these junctions coexist.     

Scattering of MCF7 Cells by Heregulin ?-1 Depends on the MEK and p38 MAP Kinase Pathway

Heregulin (HRG) β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell–cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell–cell adhesion.     

Organization of multiprotein complexes at cell–cell junctions

The formation of stable cell-cell contacts is required for the generation of barrier-forming sheets of epithelial and endothelial cells. During various physiological processes like tissue development, wound healing or tumorigenesis, cellular junctions are reorganized to allow the release or the incorporation of individual cells. Cell-cell contact formation is regulated by multiprotein complexes which are localized at specific structures along the lateral cell junctions like the tight junctions and adherens junctions and which are targeted to these site through their association with cell adhesion molecules. Recent evidence indicates that several major protein complexes exist which have distinct functions during junction formation. However, this evidence also indicates that their composition is dynamic and subject to changes depending on the state of junction maturation. Thus, cell-cell contact formation and integrity is regulated by a complex network of protein complexes. Imbalancing this network by oncogenic proteins or pathogens results in barrier breakdown and eventually in cancer. Here, I will review the molecular organization of the major multiprotein complexes at junctions of epithelial cells and discuss their function in cell-cell contact formation and maintenance.     

Dynamic cross-talk between cells and the extracellular matrix in the testis

In the seminiferous tubule of the mammalian testis, one type A1 spermatogonium (diploid, 2n) divides and differentiates into 256 spermatozoa (haploid, n) during spermatogenesis. To complete spermatogenesis and produce approximately 150 x 10(6) spermatozoa each day in a healthy man, germ cells must migrate progressively across the seminiferous epithelium yet remain attach to the nourishing Sertoli cells. This active cell migration process involves precisely controlled restructuring events at the tight (TJ) and anchoring junctions at the cell-cell interface. While the hormonal events that regulate spermatogenesis by follicle-stimulating hormone and testosterone from the pituitary gland and Leydig cells, respectively, are known, less is known about the mechanism(s) that regulates junction restructuring during germ cell movement in the seminiferous epithelium. The relative position of tight (TJs) and anchoring junctions in the testis is of interest. Sertoli cell TJs that constitute the blood-testis barrier (BTB) are present side by side with anchoring junctions and are adjacent to the basement membrane. This intimate physical association with the TJs, the anchoring junctions and the basement membrane (a modified form of extracellular matrix, ECM) suggests a role for the ECM in the junction dynamics of the testis. Indeed, evidence is accumulating that ECM proteins are crucial to Sertoli cell TJ dynamics. In this review, we discuss the pivotal role of tumor necrosis factor alpha (TNFalpha) on BTB dynamics via its effects on the homeostasis of ECM proteins. In addition, discussion will also be focused on the novel findings regarding the role of non-basement-membrane-associated ECM proteins and components of focal adhesion (a cell-matrix anchoring junction type) in the regulation of junction dynamics in the testis.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Attaching and effacing pathogen-induced tight junction disruption in vivo

Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen-infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild-type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption.     

The gastric mucosal barrier: structure of intercellular junctions in the dog

The canine gastric mucosa consists of two regions, the surface mucous cells and gland area cells including parietal, chief, and mucous-containing cells. We have used quantitative freeze-fracture methods in conjunction with thin-section extracellular tracers to document and correlate tight junction morphology with epithelial permeability. The number of strands in the tight junction complexes of the surface cells and gland cells is the same, but differences in strand arrangement exist. The surface cells have an interwoven tight junction configuration which is impermeable to extracellular tracers. The gland cell junctions are regularly arranged and often permeable to extracellular lanthanum. The possibility that the observed difference in permeability between the tight junctions of the surface mucous cells and those of the gland cells is related to their structural configuration is discussed.     

THE STRUCTURAL ORGANIZATION OF THE SEPTATE AND GAP JUNCTIONS OF HYDRA

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50–60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80–100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15–0.5 µ in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.     

Two Types of Functionally Distinct Fiber Containing Structural Protein Complexes Are Produced during Infection of Adenovirus Serotype 5

Adenoviruses are common pathogens. The localization of their receptors coxsackievirus and adenovirus receptor, and desmoglein-2 in cell-cell junction complexes between polarized epithelial cells represents a major challenge for adenovirus infection from the apical surface. Structural proteins including hexon, penton base and fiber are excessively produced in serotype 5 adenovirus (Ad5)-infected cells. We have characterized the composition of structural protein complexes released from Ad5 infected cells and their capacity in remodeling cell-cell junction complexes. Using T84 cells as a model for polarized epithelium, we have studied the effect of Ad5 structural protein complexes in remodeling cell-cell junctions in polarized epithelium. The initial Ad5 infection in T84 cell culture was inefficient. However, progressive distortion of cell-cell junction in association with fiber release was evident during progression of Ad5 infection. Incubation of T84 cell cultures with virion-free supernatant from Ad5 infected culture resulted in distortion of cell-cell junctions and decreased infectivity of Ad5-GFP vector. We used gel filtration chromatography to fractionate fiber containing virion–free supernatant from Ad5 infected culture supernatant. Fiber containing fractions were further characterized for their capacity to inhibit the infection of Ad5-GFP vector, their composition in adenovirus structural proteins using western blot and LC-MS/MS and their capacity in remolding cell-cell junctions. Fiber molecules in complexes containing penton base and hexon, or mainly hexon were identified. Only the fiber complexes with relatively high content of penton base, but not the fiber-hexon complexes with low penton base, were able to penetrate into T84 cells and cause distortion of cell-cell junctions. Our findings suggest that these two types of fiber complexes may play different roles in adenoviral infection.