Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.     

Morphologic characteristics of intercellular junctions in early embryogenesis of mice

Dynamics concerning certain intercellular junctions have been followed during the preimplantation period of development in mouse embryos. The morphological analysis of the preimplantational embryos has demonstrated, that at the initial stages of cleavage (2-4 blastomeres) the cells make contacts by means of nonspecific junctions. Specialized intercellular junctions appear at the stage of 8 blastomeres and are presented as dotted tight and gap junctions. When the embryo is developing from the stage of 8 up to the stage of 16 blastomeres, certain connective complexes appear, consisting of dotted or cord-like tight and gap junctions. At the late morula stage, the external blastomeres in the apical part have contacts with each other by means of cingular tight junctions. In this place a connective complex might emerge; it is displayed as a tight junction and one or two gap junctions. At the blastocyst stage desmosomes and adhision zones appear. Between trophectodermal cells a connective complex arises; it is presented in the slice as a tight cingular junction, desmosomes (as a rule two) and an adhision zone. Between cells of the internal cellular mass the intercellular junctions are presented as dotted tight and gap junctions. Cells of the polar trophoectoderm and cells of the internal cellular mass could have contacts by means of gap and dotted tight junctions.     

Morphogenesis in cell colonies grown from Convolvulus cell suspensions plated on synthetic media

Filtered cell suspensions of cultured callus tissue derived from the roots of Convolvulus arvensis L. were plated out on synthetic agar nutrient media in petri plates. Cell colonies which formed from the single cells or small cell groups in the suspension showed a considerable range of developmental patterns depending upon the physical and chemical environment to which they were exposed. Variation of the auxin and kinin concentrations and the nature and concentration of the source of reduced N compounds had the most profound effects on colony development. High auxin favored cell enlargement, high kinin favored the development of compact colonies composed of many small cells. Both auxin and kinin were required for cell colony formation. Cell differentiation responses which were observed but not subject to experimental control included formation of starch- and crystal-storing cells, differentiation of tracheary elements, formation of cellular filaments, and development of chlorophyllous tissue. Organ initiation was studied in cell colonies developed directly from plated cell suspensions and in cell colonies subcultured on various nutrient media. Bud initiation was produced repeatedly on media containing NAA at 10^-8 to 10^-6 m combined with kinetin at 10^-6 m . Root initiation was induced infrequently and unpredictably. Once roots had been formed from cell colonies derived from cell suspensions, the roots could be subcultured and induced to form buds; these in turn grew into whole plants. Subculture of young cell colonies to media containing different combinations of growth substances made possible a study of the effects of auxin and kinin on organization of primordia by the cell colonies. By following marked single cells plated on synthetic media, it was possible to produce single-cell clones which under proper nutrient conditions were induced to form buds. The value of the combined techniques of cell suspension culture and cell plating for the study of the physical and chemical factors influencing cell differentiation and organized development are pointed out.     

The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium

Studies of the developing trophectoderm in the mouse embryo have shown that extensive cellular remodeling occurs during epithelial formation. In this investigation, confocal immunofluorescence microscopy is used to examine the three-dimensional changes in cellular architecture that take place during the polarization of a terminally differentiated epithelial cell line. Madin-Darby canine kidney cells were plated at a low density on permeable filter supports. Antibodies that specifically recognize components of the tight junction, adherens junction, microtubules, centrosomes, and the Golgi complex were used to study the spatial remodeling of the cytoarchitecture during the formation of the polarized cell layer. The immunofluorescence data were correlated with establishment of functional tight junctions as measured by transepithelial resistance and back-exchange of the cell surface, labeled with metabolites of the fluorescent lipid analogue N-(7-[4- nitrobenzo-2-oxa-1,3-diazole]) aminocaproyl sphingosine. 1 d after plating, single cells had microtubules, radiating from a broad region, that contained the centrosomes and the Golgi complex. 2 d after plating, the cells had grown to confluence and had formed functional tight junctions close to the substratum. The centrioles had split and no longer organized the microtubules which were running above and below the nucleus. The Golgi complex had spread around the nucleus. By the fifth day after plating, the final polarized state had been achieved. The junctional complex had moved greater than 10 microns upward from its basal location. The centrioles were together below the apical membrane, and the Golgi complex formed a ribbon-like convoluted structure located in the apical region above the nucleus. The microtubules were organized in an apical web and in longitudinal microtubule bundles in the apical-basal axis of the columnar cell. The longitudinal microtubules were arranged with their minus ends spread over the apical region of the cell and their plus ends toward the basal region. These findings show that there is an extensive remodeling of epithelial cytoarchitecture after formation of cell-cell contacts. Reorganization of the microtubule network results in functional polarization of the cytoplasm.     

Access and distribution of exogenous substances in the intercellular clefts of the rat adenohypophysis

In model experiments with the use of horseradish peroxidase (HRP), two pathways of transport of substances to the adenohypophysis were studied, as well as the distribution of the tracer in the latter organ. The first pathway allows the tracer to penetrate from the intercellular milieu of the median eminence below the meningeal sheath covering the adenohypophysis to the surface of the pituitary gland. The second pathway transports the tracer via the capillaries of the hypophysial portal circulation to the interior of the glandular parenchyma. These results show (i) that the meningeal sheath establishes a barrier between the hemal milieu of the pituitary and the hemal milieu of the general circulation, and (ii) that the tracer reaching the adenohypophysis via both routes is found in the intercellular clefts of the glandular parenchyma only to a limited extent. By means of conventional electron microscopy, intercellular contacts between hormone-producing adenohypophysial cells are observed resembling focal tight junctions. Between the membranes of entwined processes of stellate cells, only small maculae adhaerentes are found. Freeze-etch studies on unfixed adenohypophyses reveal zonulae occludentes between the durafacing layers of the meningeal sheath and focal maculae occludentes between parenchymal cells. Additional tissue-culture experiments with adenohypophysial cells directly exposed to HRP reveal a gradual cessation of the labeling process in the intercellular clefts in accord with the observations from the in-vivo experiments, as well as intercellular focal tight junctions between individual hormone-producing cells.     

Endothelial cell motility is compatible with junctional integrity

Confluent endothelial cells in culture are generally regarded as a model of resting endothelium in blood vessels (i.e., forming junctions at points of cell-cell contact, losing ability to proliferate in response to growth factors, and remaining stationary). However, incompatibility between junctional integrity and endothelial cell motility remains uncertain. The aim of this study was to determine whether endothelial cells (in colonies generated from differentiating embryonic stem cells in contact with OP9 stromal cell layer) have a resting endothelial phenotype (i.e., lack motility). Time-lapse analyses showed that though endothelial cells were connected to each other through adherens junctions and tight junctions, they were moving continuously within the colonies. Endothelial cell movement was accompanied by formation of lamellipodia, which transiently accumulated green fluorescent protein-tagged beta-actin and p41-Arc (a subunit of the actin-related protein 2/3 complex) at their anterior tips, suggesting that the movement is an active behavior of endothelial cells. Endothelial cell-specific expression of yellow fluorescent protein-tagged vascular endothelial-cadherin and claudin-5 revealed that adherens junctions and tight junctions persisted during endothelial cell migration. Furthermore, intercellular junctions underwent dynamic remodeling at the leading edge of moving endothelial cells. These results suggest that endothelial cells can remain highly motile without losing intercellular junctions.     

The ultrastructure of the rat primary decidual zone

The rat primary decidual zone (PDZ) is a transitory, avascular region of transformed fibroblasts surrounding the implanting embryo. Tracer studies have indicated that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight of the tracer. To clarify the morphological basis of the permeability barrier, we have studied the ultrastructure of the PDZ with particular emphasis on the intercellular features and cellular junctions. The cells of the PDZ were large and tightly packed; their apposed membranes showed extensive interdigitations in some regions, but elsewhere they were relatively straight. Tight junctions, gap junctions, and desmosomelike junctions were observed between decidual cells. The tight junctions usually consisted of one or two points of membrane fusion, and they were oriented both parallel and perpendicular to the long axis of the PDZ. These junctions were frequently associated with gap junctions. Scattered pockets of dilated extracellular space between decidual cells contained collagen fibrils and an amorphous, dense material. These extracellular components were also sequestered by the decidual cells in deep invaginations of the cell surface that were continuous with the extracellular space. Decidual cells also exhibited flangelike processes that penetrated the basal laminae of the adjacent epithelium and capillary endothelium. Our present observations indicate that decidual cells are connected by tight junctions, and a previous study demonstrated that macromolecules up to 40 kDa readily cross the PDZ; hence, the tight junctions appear to be discontinuous. We suggest that the structures restricting the movement of large macromolecules (66 kDa and larger) across the PDZ from blood vessels to the embryo may include discontinuous tight junctions, membrane interdigitations, and amorphous intercellular material.     

Lipid-protein cargo transfer: a mode of direct cell-to-cell communication for lipids and their associated proteins

Cells in tissues or in experimental cell colonies respond to stimuli in a co-ordinated manner when they are electrically and chemically coupled by gap junctions. These junctions permit the cell-to-cell passage of small molecules, such as inositol tris phosphate (IP(3)) within the colony and are important in co-ordinating tissue activity. This is the only recognised mechanism of direct chemical signalling that does not involve the release of an extracellular messenger between cells. However, the data in this article demonstrates a new mode of intercellular communication. Two potentially important signalling lipids, PIP(2) and ganglioside G-M1 were shown to move between cells in colonies by tracking (i) fluorescent lipids loaded into the plasma membranes of individual cells in a cell colony using a novel micropipette technique and (ii) movement of fluorescent lipids after localised photobleaching. Furthermore, a large protein molecule, cholera toxin B subunit bound to extracellularly facing ganglioside G-M1 was also shown to transfer between cells. The transfer was inhibited by pre-treatment with poly-L-lysine and polyethylenimine, suggesting a role for tight junctions, perhaps by permitting diffusion of lipids and their protein "cargo" across these cell-to-cell contact points. This is a hitherto unsuspected form of molecular signalling within cell colonies and tissues which may have implications for understanding co-ordinated cell colony behaviour.     

Disentangling cadherin-mediated cell-cell interactions in collective cancer cell migration

Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single-cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration.     

Electrical, contractile, and ultrastructural properties of adult rat and guinea-pig ventricular myocytes in long-term primary cultures

The electrical, contractile, and morphological characteristics of ventricular myocytes isolated from adult rat and guinea-pig hearts and maintained in cultures for 7-24 days are described. These cultured cells form different networks, depending on the species, when plated at certain density and maintained under specific conditions; the cells within the networks appear to be electrically coupled. Their resting and action potentials, their contractile activity (shortenings), and their pharmacological responses qualitatively resemble those of freshly isolated myocytes. Cultured cells from both species exhibit near-normal ultrastructural organization of sarcomeres, myofilaments, and mitochondria, as well as formation of intercellular contacts, or gap junctions. These data indicate that cultured adult rat and guinea-pig myocardial cells that make intercellular contacts possess electrical, contractile, and ultrastructural properties and responses to pharmacological agents similar to those of the respective adult myocardial tissues and the functionally intact freshly isolated cells from which these cultures are prepared. Thus, this study indicates that long-term cultures (7-24 days) of networked cardiac myocytes could be used as a valuable experimental model in various investigations of excitation-contraction coupling in cardiac muscle.     

The cellular basis of self renewal in culture by human acute myeloblastic leukemia blast cell progenitors

Blast cells from patients with Acute Myeloblastic Leukemia (AML) were separated according to cell size using velocity sedimentation under unit gravity. Fractions obtained in this way were plated in methyl cellulose with a growth stimulator present in media conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM). Colonies of blast cells form under these conditions. Pooled cell suspensions from such colonies were plated in microwells; the plating efficiency of such suspensions is a measure of blast progenitor self-renewal occurring in the original blast colonies. Self-renewal assays on each fraction indicated that self renewal among blast progenitors is heterogeneously distributed with subpopulations differing in renewal capacities. The results are consistent with the view that blast cell subpopulations in AML undergo a series of transitions associated with decreasing self renewal capacity, analogous to that observed in normal hemopoiesis, where proliferative capacity decreases with increasing differentiation.     

The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions.

Adherens-type junctions (AJs) are major subcellular targets for tyrosine specific protein phosphorylation [Volberg et al. (1991) Cell Regul., 2, 105-120]. Here we report on the apparent effect of such phosphorylation events on the assembly and integrity of AJs. We show that incubation of MDCK cells with potent inhibitors of tyrosine-specific phosphatases (PTP), namely H2O2 and vanadate, leads to a dramatic increase in AJ-associated phosphotyrosine which was apparent already within 2-5 min of treatment and progressed upon further incubation. Examination of H2O2 vanadate treated cells at later time points indicated that intercellular AJs rapidly deteriorated, concomitantly with a marked increase in the number and size of vinculin and actin containing focal contacts. In parallel, major changes were observed in cell structure and topology, as revealed by electron microscopy. These were manifested by rapid rounding-up of the cells followed by reorganization of the cell monolayer. Other intercellular junctions, including desmosomes and tight junctions, visualized by staining with desmoplakin and ZO-I antibodies, were not significantly affected. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we have carried out reciprocal experiments in which Rovs Sarcoma virus (RSV) transformed chick lens cells, expressing high levels of pp60src kinase, were treated with inhibitors of tyrosine kinases, (tyrphostins). We show that following such treatment, intercellular AJs which were deteriorated in the transformed cells, were reformed. Based on these observations, we propose that specific tyrosine phosphorylation of AJ components is involved in the downregulation of these cellular contacts.     

Functional analysis of tight junctions

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.     

Mechanical plasticity in collective cell migration

Collective cell migration is crucial to maintain epithelium integrity during developmental and repair processes. It requires a tight regulation of mechanical coordination between neighboring cells. This coordination embraces different features including mechanical self-propulsion of individual cells within cellular colonies and large-scale force transmission through cell–cell junctions. This review discusses how the plasticity of biomechanical interactions at cell–cell contacts could help cellular systems to perform coordinated motions and adapt to the properties of the external environment.     

Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing

Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell–cell contact plays in this phenomenon. The formation of cell–cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca²⁺ restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell–cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing. © 1994 Wiley-Liss, Inc.     

Cell-to-cell contacts between lymphoreticular cells in rheumatoid synovial membrane

Nodular type and diffuse perivascular lymphoid cell infiltrates in rheumatoid synovial membranes from 72 patients were examined under the electron microscope. Attempts were made to describe the ultrastructural relations of the cells examined and to classify the different types of cell-to-cell contracts between them. Members of a complex series, composed of gradually differentiating forms of cell contacts beginning from simple membrane adherences and interdigitations and gap junctions; minute filamentous intercellular bridges: subsurface confronting cisternae and desmosome-like structures, are described and suggested as function-dependent morphological representatives of sites of cellular interactions.     

Intercellular junctions between human odontoblasts. A freeze-fracture study after demineralization

Intercellular junctions in the odontoblastic layer have been studied with a freeze-fracture technique. Children's tooth germs were fixed, sliced and demineralized. Samples of the pulpodentinal border were routinely prepared for freeze-fracture. Three kinds of intercellular junctions were detected between human odontoblast cell bodies: gap junctions, desmosomes and tight junctions. Numerous gap junctions are responsible for intercellular communication at different levels of the cell bodies. Focal tight junctions, parallel to the axis of the cell, and desmosomes are sites of cell-to-cell adhesion between lateral plasma membranes. At the distal end of the cell bodies, junctional complexes consist of zonular tight junctions and gap junctions. These zonular tight junctions, never before described between odontoblasts, contribute to the pseudo-epithelial organization of the odontoblastic layer. They constitute a predentin-pulp barrier, the permeability of which must be studied to establish their role in relation to dentin formation.     

Junctions between pericytes and the endothelium in rat myocardial capillaries: A morphometric and immunogold study

Summary Pericytes are cells of mesodermal origin which are closely associated with the microvasculature. Despite numerous studies little is known about their function. We have studied the relationship between pericytes and the endothelium in rat myocardial capillaries employing ultrastructural and immunogold techniques. 14% of the subendothelial cell membrane is covered by comparatively small pericytic cell processes. About half of these processes are completely embedded in baseement membrane material, whereas the remaining half forms closer contacts with the endothelium. These contacts are devoid of anti-laminin immunogold label, a marker for basement membranes. A small fraction of these contacts has been identified as tight junctions resembling those seen between endothelial cells in capillaries of the same tissue. The remaining majority of junctions reveals a cleft of approximately 18 nm between the apposed membranes in which a succession of cleft-spanning structures can often bedetected. It was also found that pericytic processes are preferentially located close to interendothelial junctions. We suggest that the high frequency of intimate junctions between pericytes and the endothelium and the preferential localisation near paracellular clefts may have functional significance.     

Enrichment of progenitor cells from human bone marrow by flow cytometry

Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean ± standard deviation) with respect to the total was as follows: Region 1: 17.6±9.9; region 2: 5.3±1.4; region 3: 71.7±9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.