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PubMed1991

Desmosome biogenesis in the mouse preimplantation embryo.

The molecular processes underlying the formation of the first desmosomes in the mouse early embryo have been examined by immunocytochemical and biochemical techniques using antibody probes recognising desmosomal proteins 1 and 2 (dp1 + 2, desmoplakins), dp3 (plakoglobin), desmosomal glycoprotein 1 (dg1, desmoglein) and dg2 + 3 (desmocollins). Immunofluorescence labelling of staged intact embryos and synchronised cell clusters indicates that dp1 + 2, dg1 and dg2 + 3 are first detectable on the lateral membrane contact sites between trophectoderm cells in early cavitating blastocysts, coincident with the onset of desmosome formation as seen in ultrastructural preparations. Membrane localisation of these antigens is predominantly punctate in appearance, occurs after division to the 32-cell stage and appears to be coincident with blastocoele formation since non-cavitated embryos/cell clusters of equivalent age/cell cycle are usually unlabelled. In contrast, dp3 is first detectable at the 32-cell stage at all internal membrane contact sites (including those with inner cell mass cells) in a continuous linear pattern, and appears in both cavitated and non-cavitated specimens. Subsequently during blastocyst expansion, dp3 localisation becomes punctate and restricted to trophectodermal membranes. Immunoprecipitation of desmosomal antigens following metabolic labelling indicates that synthesis of dp3 is underway from at least compaction in the 8-cell embryo, while dp1 + 2 synthesis is first evident in 16-cell morulae. Synthesis of dg1 and dg2 + 3 is not detectable until the early blastocyst stage. These results suggest that desmosome biogenesis in the preimplantation embryo might be regulated by transcription or translation of desmosomal glycoproteins and by maturational changes in the trophectoderm layer associated with blastocoele formation. The earlier expression and wider distribution of dp3 at cell contact areas may reflect non-desmosomal sites (eg, adherens junctions) for this protein and a possible role for dp3 in the development of intercellular junctions.

Animals↗
PubMed2004

The specific fates of tight junction proteins in apoptotic epithelial cells.

The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped C-terminally to Asp(320) within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells.

Animals↗
PubMed2003

Signal transduction of gap junctional genes, connexin32, connexin43 in human hepatocarcinogenesis.

AIM: To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis. METHODS: Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot. RESULTS: SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein. CONCLUSION: The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.

Calcium Signaling↗
PubMed1993

Ultrastructure of invasion of the adult rat hepatocyte monolayer by rat ascites hepatoma variant sublines.

Invasion of a primary cultured adult rat hepatocyte monolayer by rat ascites hepatoma AH7974-derived variant sublines (74AD, 74FL) was studied ultrastructurally. Although 74AD and 74FL are an adherent and a floating subline, respectively, in ordinary in vitro culture, the majority of the cells of both sublines attached to the hepatocytes at the tips of their cytoplasmic processes up to 2 h after coculture. Thereafter, these cells adhered to the hepatocytes with several types of junctional structures. Simple apposition was prominent 6 h after coculture, and intermediate and tight junctions were frequently observed 12 h after coculture. The attached tumor cells separated the intercellular junctions of the hepatocyte monolayer with their cytoplasmic protrusions and became directly adherent to dishes by pushing aside the hepatocytes. In addition, we sometimes found some peculiar interactions which appeared to be an early event of a kind of cytoplasmic fusion between a tumor cell and a hepatocyte after 12 h of coculture. An increasing electron density of mitochondria, the disappearance of cristae, and the presence of cytoplasmic vacuolations were observed in the hepatocytes fused with tumor cells. Such results suggest that the hepatic invasion by tumor cells results from the interaction of tumor cells and hepatocytes leading to their cytoplasmic fusion, as well as to direct migration of tumor cells into hepatic cords. The mechanism of invasion may be independent of that of substrate adhesion.

Animals↗
PubMed1997

Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners.

Since macromolecular permeability between endothelial cells is regulated by tight junctions (zonula occludens), we wished to determine whether they also regulate neutrophil transendothelial migration. HUVEC monolayers, a commonly used model for studying leukocyte transmigration, were characterized using electric cell substrate impedance sensing and transmission electron microscopy. We show that culture medium containing endothelial cell growth supplement (50 microg/ml) was sufficient and necessary for the development of endothelial tight junctions. The frequency with which tight junctions were observed by transmission electron microscopy was further increased (twofold) by culturing HUVEC monolayers in a 1:1 mixture of endothelial medium and astrocyte-conditioned medium. These astrocyte-conditioned HUVEC monolayers showed a >1.5-fold increase in transcellular electrical resistance. The extent of neutrophil migration across IL-1-treated (10 U/ml for 4 h) HUVEC monolayers was the same whether tight junctions were present or absent, and the molecular requirements for neutrophil transmigration (CD18 and intercellular adhesion molecule-1) were unaffected by culturing in astrocyte-conditioned medium. Immunostaining for proteins associated with the intercellular junctional domain (occludin, ZO-1, cadherin, beta-catenin, gamma-catenin, and platelet-endothelial cell adhesion molecule-1) was localized to the endothelial borders, regardless of the culture conditions. Discontinuities were observed in the border staining for occludin, ZO-1, cadherin, and beta-catenin at the tricellular corner where the borders of three endothelial cells intersected. Significantly, 75% of neutrophil migration across IL-1-treated HUVEC monolayers occurred at tricellular corners. It appears that neutrophils preferentially migrate around endothelial tight junctions by crossing at tricellular corners rather than passing through the tight junctions that lie between two endothelial cells.

Astrocytes↗
PubMed2006

Disruption of tight junctions and induction of proinflammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni.

Campylobacter jejuni is a leading cause of human enterocolitis and is associated with postinfectious complications, including irritable bowel syndrome and Guillain-Barré syndrome. However, the pathogenesis of C. jejuni infection remains poorly understood. Paracellular pathways in intestinal epithelial cells are gated by intercellular junctions (tight junctions and adherens junctions), providing a functional barrier between luminal microbes and host immune cells in the lamina propria. Here we describe alterations in tight junctions in intestinal epithelial monolayers following C. jejuni infection. Apical infection of polarized T84 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TER). Immunofluorescence microscopy revealed a redistribution of the tight junctional transmembrane protein occludin from an intercellular to an intracellular location. Subcellular fractionation using equilibrium sucrose density gradients demonstrated decreased hyperphosphorylated occludin in lipid rafts, Triton X-100-soluble fractions, and the Triton X-100-insoluble pellet following apical infection. Apical infection with C. jejuni also caused rapid activation of NF-kappaB and AP-1, phosphorylation of extracellular signal-regulated kinase, Jun N-terminal protein kinase, and p38 mitogen-activated protein kinases, and basolateral secretion of the CXC chemokine interleukin-8 (IL-8). Basolateral infection with C. jejuni caused a more rapid decrease in TER, comparable redistribution of tight-junction proteins, and secretion of more IL-8 than that seen with apical infection. These results suggest that compromised barrier function and increased chemokine expression contribute to the pathogenesis of C. jejuni-induced enterocolitis.

Campylobacter jejuni↗
PubMed2006

Filamin A (FLNA) is required for cell-cell contact in vascular development and cardiac morphogenesis.

Mutations in the human Filamin A (FLNA) gene disrupt neuronal migration to the cerebral cortex and cause cardiovascular defects. Complete loss of Flna in mice results in embryonic lethality with severe cardiac structural defects involving ventricles, atria, and outflow tracts, as well as widespread aberrant vascular patterning. Despite these widespread developmental defects, migration and motility of many cell types does not appear to be affected. Instead, Flna-null embryos display abnormal epithelial and endothelial organization and aberrant adherens junctions in developing blood vessels, heart, brain, and other tissues. Essential roles for FLNA in intercellular junctions provide a mechanism for the diverse developmental defects seen in patients with FLNA mutations.

Animals↗
PubMed1998

Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells.

The cytoplasmic calcium environments along membrane trafficking pathways leading to gap junction intercellular communication channels at the plasma membrane were studied. Connexins, the constitutive proteins of gap junctions, were fused at their carboxyl terminus to the calcium-sensitive photoprotein aequorin. The cellular location of the chimeric proteins was determined by immunolocalization and subcellular fractionation. The generation of functional gap junctions by the connexin chimerae was monitored by the ability of the cells to exchange small dyes. Although aequorin fused to connexin-26 was nonfunctional, its ability to report Ca2+ and to form functional gap junctions was rescued by replacement of its cytoplasmic carboxyl tail with that of connexin-43. In COS-7 cells expressing these connexin-aequorin chimerae, calcium levels below the plasma membrane were higher (approximately 5 microM) than those in the cytoplasm (approximately 100 nM); gap junctions were able to transfer dyes under these conditions. Cytoplasmic levels of free calcium surrounding the ERGIC/Golgi reported by connexin-43 chimera (approximately 420 nM) were twice those measured by connexin-32 chimera (approximately 200 nM); both chimerae measured calcium levels substantially higher than those reported by a connexin-26 chimera (approximately 130 nM). Dispersion of the ERGIC and Golgi complex by brefeldin A led to a marked reduction in calcium levels. The results show that the various connexin chimerae were located in spatially different subcellular stores and that the ERGIC/Golgi regions of the cell maintain heterogeneous cytoplasmic domains of calcium. The implications of the subplasma-membrane Ca2+ levels on the gating of gap junctions are discussed.

Aequorin↗
PubMed2006

Low conductance gap junctions mediate specific electrical coupling in body-wall muscle cells of Caenorhabditis elegans.

Invertebrate innexins and their mammalian homologues, the pannexins, are gap junction proteins. Although a large number of such proteins have been identified, few of the gap junctions that they form have been characterized to provide combined information of biophysical properties, coupling pattern, and molecular compositions. We adapted the dual whole cell voltage clamp technique to in situ analysis of electrical coupling in Caenorhabditis elegans body-wall muscle. We found that body-wall muscle cells were electrically coupled in a highly organized and specific pattern. The coupling was characterized by small (350 pS or less) junctional conductance (G(j)), which showed a bell-shaped relationship with junctional potential (V(j)) but was independent of membrane potential (V(m)). Injection of currents comparable to the junctional current (I(j)) into body-wall muscle cells caused significant depolarization, suggesting important functional relevance. The innexin UNC-9 appeared to be a key component of the gap junctions. Both Myc- and green fluorescent protein-tagged UNC-9 was localized to muscle intercellular junctions. G(j) was greatly inhibited in unc-9(fc16), a putative null mutant. Specific inhibition of UNC-9 function in muscle cells reduced locomotion velocity. Despite UNC-9 expression in both motor neurons and body-wall muscle cells, analyses of miniature and evoked postsynaptic currents in the unc-9 mutant showed normal neuromuscular transmission. These analyses provide a relatively detailed description of innexin-based gap junctions in a native tissue and suggest that innexin-based small conductance gap junctions can play an important role in processes such as locomotion.

Animals↗
PubMed1998

The tight junction: morphology to molecules.

The tight junction forms a regulated barrier in the paracellular pathway between epithelial and endothelial cells. This intercellular junction also demarcates the compositionally distinct apical and basolateral membranes. While the existence of a paracellular barrier in epithelia was hypothesized by physiologists over a century ago, the molecular characterization of the tight junction is a relatively new and rapidly expanding area of research. It is now recognized that the tight junction is comprised of at least nine peripheral and one integral membrane proteins. This complex includes members of a protein family related to tumor suppression and signal transduction, a rab protein, and a Ras target protein. The characteristics of, interactions between, and potential physiological roles of these proteins at the tight junction are discussed.

Animals↗
PubMed1978

The fine structure of ependyma in the hypothalamus of the hamster (Cricetus cricetus).

The ciliated ependyma of the hypothalamus of the hanster (Cricetus cricetus) consists of a sheet of cells, the luminal surface of which is reflected over cilia and numerous microvilli. The lateral portions of the plasmalemma of contiguous cells are fused at some sites to from junctions of five layers, the zonula occludens. These fusions occur usually in continuity with other intercellular junctions, the zonula adhaerens. In the apical regions of the ependyma these fusions are almost spiralled. The nucleus is regularly oval and lying close to the basal plasmalemma. The cytoplasm contains a finely granular matrix, filamentous components close to the junctions. The endoplasmic reticulum is sparse. The Golgicomplex is confined to the supranuclear cytoplasm, and mitochondria, are more numerous in the apical than in the basal regions of ependymal cells; the mitochondria are generally rounded and have transverse cristae and granules. The apical regions of the cytoplasm contain the basal bodies of cilia; they form non-striated rootlets and the rootlet filaments tend to diverge, ending in a granular zone.

Animals↗
PubMed2002

Functional significance of gap junctional coupling in preimplantation development.

Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or beta6, connexin 36 or alpha9, and connexin 57 or alpha10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18alpha-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Animals↗
PubMed2005

Alterations in connexin26 expression during colorectal carcinogenesis.

BACKGROUND: Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions, which consist of transmembrane proteins called connexins (Cxs). Human colorectal epithelial cells express Cx32 and Cx43. METHODS: Tissue samples (152 from colorectal cancer and 75 from adenoma) were investigated by immunohistochemistry, using the antibody for Cx26. Moreover, Cx26 was assessed in normal epithelium of the colon and rectum, adjacent to colorectal cancer. RESULTS: In normal epithelium and adenomas, intercellular, punctate staining for Cx26 was observed. In adenomas with severe dysplasia, focally decreased expression of Cx26 was observed. Among 152 colorectal cancers, 55.9% classified only as adenocarcinoma stained positive for Cx26, but mainly cytoplasmic staining was found. We observed a positive correlation between Cx26 expression and tumor G2 grade (p < 0.005). The expression of Cx26 did not correlate with age, sex of patients, tumor localization, lymph node status or tumor size. CONCLUSIONS: Our results show that during colorectal carcinogenesis, loss of normal intercellular Cx expression occurs. Furthermore, the cytoplasmic presence of Cx26 could indicate a different role of Cx26 in neoplastic cells than participation in GJIC.

Adult↗
PubMed2003

Developmental regulation of gap junctions and their role in mammary epithelial cell differentiation.

Gap junctions play a critical role in the development and differentiation of many tissues. Formed by the joining of two connexons on opposing membranes of two cells, gap junctions permit passage of ions and small molecules. Six connexins (Cx) belonging to a family of closely related tetraspan transmembrane proteins form a connexon. Connexin expression peaks in lactation, and those identified in the gland, thus far, are Cx26, Cx30, Cx32, and Cx43. Cx43 associates with myoepithelial cells, while others associate with epithelial and ductular cells. In vitro, assembly of functional gap junctions appears to be essential for differentiation of mammary epithelial cells. However, the role of gap junction intercellular communication (GJIC) in differentiation and growth remains unclear. Recent evidence challenges the view that gap junctions are simply pore-forming proteins and suggests that cell adhesion-associated proteins interact with the cytosolic carboxy-terminus of connexins and participate in signaling events. The possible implications on mammary cell function are discussed.

Animals↗
PubMed1999

Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo.

In the present paper, we characterize an antibody, mAb BV13, directed to mouse vascular endothelial (VE)-cadherin, a major adhesive protein of interendothelial adherens junctions. When added to cultured endothelial cells, BV13 induces a redistribution of VE-cadherin from intercellular junctions. VE-cadherin redistribution did not change the localization of platelet endothelial cell adhesion molecule or tight junction markers such as zonula occludens 1, cingulin, and junctional adhesion molecule. Intravenous administration of mAb BV13 induced a concentration- and time-dependent increase in vascular permeability in heart and lungs. By electron microscopy, interstitial edema and accumulation of mixed types of inflammatory cells in heart and lungs were observed. Injection of (rhodamine-labeled) Ricinus communis I lectin showed focal spots of exposed basement membrane in the alveolar capillaries and in some larger pulmonary vessels. These data indicate that VE-cadherin is required for vascular integrity and normal organ functions.

Animals↗
PubMed2001

Gap junctions and fluid flow response in MC3T3-E1 cells.

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca(2+) concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E(2) (PGE(2)) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca(2+) (Ca) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE(2) but not to the Ca response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

Animals↗
PubMed1977

Left atrial ultrastructure in mitral valvular disease.

Light microscopic and ultrastructural observations were made on left atrial tissues obtained from 14 patients at the time of operation for correction of mitral valvular disease. Cardiac muscle cells varied in size but most frequently were hypertrophied. In fibrotic areas, present in all left atria, the muscle cells tended to be isolated from adjacent cells and exhibited degenerative changes of varying severity. These changes consisted or proliferation of Z-band material and cytoskeletal filaments, myofibrillar loss, proliferation of elements of free and extended junctional sarcoplasmic reticulum, variations in size and number of mitochondria, occurrence of abnormal mitochondria, dissociation of intercellular junctions, formation of spherical microparticles, and accumulation of lysosomal degradation products. Hypertrophy was considered to lead to cellular degeneration, with decrease or loss of contractile function. Atrial fibrillation was associated with severe cellular degeneration. The severity of degeneration was greater in patients with mitral regurgitation, with or without associated mitral stenosis, than in patients with pure mitral stenosis.

Adult↗
PubMed2000

Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition.

The effects of hypoxia and phosphatase inhibitors on connexin43 (Cx43) phosphorylation state, gap junctional intercellular communication (GJIC) and immunolabelling with anti-Cx43 antibodies were investigated in cultured astrocytes. Astrocytes contained predominantly phosphorylated forms of Cx43 and these underwent dephosphorylation 30 min after hypoxia. This was preceded by a 77% reduction in GJIC 15 min after hypoxia, indicating that reduced GJIC occurs prior to Cx43 dephosphorylation. Hypoxia caused a reduction in punctate immunostaining (epitope masking) at cell-cell contacts with one anti-Cx43 antibody, and increased labelling with another antibody (13-8300) that detects only a dephosphorylated form of Cx43. Inhibition of protein phosphatase (PP)-1 and PP-2A with okadaic acid or calyculin A had little effect on hypoxia-induced Cx43 dephosphorylation. Inhibition of PP-2B (calcineurin) with cyclosporin A or FK506 reduced Cx43 dephosphorylation and junctional uncoupling seen after hypoxia. These results demonstrate that responses of astrocytic Cx43 to hypoxia in vitro are similar to those seen after ischaemia in vivo, and that inhibition of protein phosphatase protects astrocytes from hypoxia-induced Cx43 dephosphorylation and junctional uncoupling. In addition, calcineurin may play a direct role in the regulation of astrocytic GJIC and Cx43 phosphorylation state.

Alkaline Phosphatase↗