Areas Of Intercellular Contact Research Articles

Editorial: Who Belongs?

The axolotl pronephric duct rudiment is readily accessible to both SEM observation and surgical manipulation. The rudiment segregates from the dorsal part of the lateral mesoderm and then extends caudally along the ventrolateral border of the segmenting comites, eventually contacting the cloacal wall. The marked thinning of the rudiment which accompanies this migration is paralleled by a corresponding reduction in cell number across the duct's diameter and by caudad translocation and elongation of vital dye marks applied to the duct mesoderm. Duct extension thus involves appreciable cell rearrangement. The morphology of duct mesoderm and its substratum (somite and lateral mesoderm) suggests that active locomotion of cells near its tip marshals the duct's caudad elongation. Filopodia and small focal areas of intercellular contact may mediate the adhesions between the cells which must be broken and reformed as the cells rearrange.

Vezatin, a protein associated to adherens junctions, is required for mouse blastocyst morphogenesis

Cell–cell interactions play a major role during preimplantation development of the mouse embryo. The formation of adherens junctions is a major feature of compaction, the first morphogenetic event that takes place at the 8-cell stage. Then, during the following two cell cycles, tight junctions form, and the outer layer of cells differentiate into a functional epithelium, leading to the formation of the blastocoel cavity. Until now, E-cadherin was the only transmembrane molecule localized in adherens junctions and required for early development. Vezatin is a transmembrane protein of adherens junctions, interacting with the E-cadherin–catenins complex. Here, we show that vezatin is expressed very early during mouse preimplantation development. It co-localizes with E-cadherin throughout development, being found all around the cell cortex before compaction and basolaterally in adherens junctions thereafter. In addition, vezatin is also detected in nuclei during most of the cell cycle. Finally, using a morpholino-oligonucleotide approach to inhibit vezatin function during preimplantation development, we observed that inhibition of vezatin synthesis leads to a cell cycle arrest with limited cell–cell interactions. This phenotype can be rescued when mRNAs coding for vezatin missing the 5′UTR are co-injected with the anti-vezatin morpholino-oligonucleotide. Cells derived from blastomeres injected with morpholino-oligonucleotide had a reduced amount of vezatin concomitantly with a decrease in the quantity of E-cadherin and β-catenin localized in the areas of intercellular contact. Shift in E-cadherin cortical distribution was correlated with a strong decrease in E-cadherin mRNA and protein contents. Altogether, these observations demonstrate that vezatin is required for morphogenesis of the preimplantation mouse embryo.

Matrix-bound fibroblasts regulate angiogenesis by modulation of VE-cadherin.

Vascular endothelial cadherin (VE-cadherin/cadherin-5) has previously been described as playing a specific role in angiogenesis due to its localisation at areas of intercellular contact, where it functions in maintenance of tubular architecture. Matrix-bound fibroblasts have been show to produce a number of factors that are important in inducing angiogenesis and to promote tubule-formation by human endothelial cells, an indicator of angiogenic potential. Tubule formation stimulated by fibroblast-derived growth factors can be prevented by the addition of monoclonal antibody to VE-cadherin. In addition, fibroblasts-derived growth factors are able to modulate the expression and hence the regulation of this endothelial cell specific cadherin. The change in VE-cadherin expression of human endothelial cells by fibroblast-derived growth factors may contribute to the regulation of angiogenesis.

VE-Cadherin Mediates Endothelial Cell Capillary Tube Formation in Fibrin and Collagen Gels1

Various cell adhesion molecules mediate the diverse functions of the vascular endothelium, such as cell adhesion, neutrophil migration, and angiogenesis. In order to identify cell adhesion molecules important for angiogenesis, we used anin vitromodel (Chalupowicz, Chowdhury, Bach, Barsigian, and Martinez,J. Cell Biol.130, 207–215, 1995) in which human umbilical vein endothelial cell monolayers are induced to form capillary-like tubes when a second gel, composed of either fibrin or collagen, is formed overlying the apical surface. In the present investigation, we observed that a monoclonal antibody directed against the first extracellular domain of human vascular endothelial cadherin (VE-cadherin, cadherin 5) inhibited the formation of capillary tubes formed between either fibrin or collagen gels. Moreover, when added to preformed capillary tubes, this antibody disrupted the capillary network. In contrast, monoclonal antibodies directed against the extracellular domain of N-cadherin, the αvβ3integrin, and PECAM-1 failed to inhibit capillary tube formation. During capillary tube formation, Western blot and RT-PCR analysis revealed no marked change in VE-cadherin expression. Immunocytochemical studies demonstrated that VE-cadherin was concentrated at intercellular junctions in multicellular capillary tubes. Thus, VE-cadherin plays a specific role in fibrin-induced or collagen-induced capillary tube formation and is localized at areas of intercellular contact where it functions to maintain the tubular architecture. Moreover, its function at tubular intercellular junctions is distinct from that at intercellular junctions present in confluent monolayers, since only the former was inhibited by monoclonal antibodies.

Steric stabilization and cell adhesion

We present theoretical and experimental arguments supporting the hypothesis that the cell surface glycocalyx may negatively regulate adhesive phenomena. First, it is recalled that a repulsive interaction of several thousands of piconewtons may be generated on a contact area of about 1/100 gm 2 by a combination of electrostatic and entropic forces (steric stabilization). Second, electron microscopical data are reported to provide an estimate of the thickness of the cell coats of murine macrophages and sheep erythrocytes made phagocytable by exposure to glutaraldehyde or specific antibodies. Using conventional carbohydrate staining procedures, it is shown that the total thickness of the electron-dark regions in areas of intercellular contact is lower than the sum of the thicknesses of electron-dark regions on free cell areas. Further, removing negative charges with neuraminidase or neutralizing these charges with polylysine may reduce intermembrane distance in contact areas. Third, it is shown that a decrease of erythrocyte surface charges with neuraminidase increases their adhesion to murine phagocytes under dynamic, not static conditions. It is concluded that a major determinant of steric stabilization is the relative length of adhesion molecules and surface repeller elements, and that repulsion is particularly important under dynamic conditions. Thus, dynamic effects must be included in models of steric stabilization.

Association of the Dictyostelium 30 kDa actin bundling protein with contact regions

'Contact regions' are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (H.M. Ingalls et al. (1986). Proc. Nat. Acad. Sci. USA 83, 4779). Purified contact regions contain a prominent actin-binding protein with an M(r) of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000 M(r) actin-bundling protein (known as 30 kDa protein), previously shown to be enriched in filopodia (M. Fechheimer (1987). J. Cell Biol. 104, 1539). About four times more 30 kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30 kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin and 30 kDa protein indicate that the 30 kDa protein is associated with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30 kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30 kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the presence of actin‐associated intercellular adhesion junctions between interstitial cells of Leydig in the ground squirrel testis

Interstitial cells of Leydig characteristically occur in clusters around blood vessels. Often these clusters remain intact when interstitial tissues are mechanically separated from other components of the testis. The presence of strong intercellular attachments is most likely one of the factors responsible for maintaining the integrity of Leydig cell clusters. In many tissues, actin associated adhesion junctions commonly provide intercellular attachment. To determine if actin associated adhesion junctions are present between Leydig cells, we have used 1) immunofluorescence to probe for two components that characterize these junctions in other tissues and 2) electron microscopy to examine areas of intercellular contact for evidence of microfilament related adhesion junctions. Isolated clusters of unsectioned cells, which had been fixed and detergent extracted, were probed with the F-actin specific strains rhodamine phalloidin and NBD-phallacidin and with an affinity purified primary antibody raised against human platelet vinculin. In regions of intercellular contact, fluorescence staining with the actin probes was intense and appeared as a solid linear band. Similar regions also stained with the vinculin probe. In double label experiments, actin and vinculin probes were co-distributed at sites of intercellular contact. Zones of intercellular contact, apparently similar to those detected with fluorescence microscopy, were observed at the ultrastructural level. At these sites, subsurface filaments, interpreted by us as actin, formed a dense carpet adjacent to the plasma membrane on each side of the junction. These filaments appeared to be organized into networks rather than discrete bundles.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation.

C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than non-transfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

Detection of Cell-CAM 105 in the pericanalicular domain of the rat hepatocyte plasma membrane

Cell-CAM 105 has been identified as a cell adhesion molecule based on the ability of anti-cell-CAM 105 monospecific Fab fragments to inhibit the reaggregation of rat hepatocytes. Because of its adhesive properties, it was expected that cell-CAM 105 would be present on the lateral cell surface where adhesive interactions predominate. Paradoxically, however, immunofluorescence analysis of frozen sections of rat liver using specific monoclonal antibodies indicated that cell-CAM 105 was present exclusively in the bile canalicular domain of the rat hepatocyte where there is no intercellular adhesion. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase labeling and electron microscopy were used to examine intact and mechanically dissociated liver tissue. Results showed that when accessibility was provided by mechanical dissociation of perfusion fixed liver tissue, cell-CAM 105 could be detected in the pericanalicular region of lateral membranes. In contrast, when hepatocytes were labeled after incubation in vitro under conditions used during adhesion assays to induce reaggregation, cell-CAM 105 rapidly redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures further revealed that cell-CAM 105 and two other bile canalicular proteins relocalized to discrete domains reminiscent of bile canaliculi, whereas cell-CAM 105 was also present in areas of intercellular contact. Serial section electron microscopy analysis of well-defined, cross-sectional profiles of bile canaliculi suggested the presence of cell-CAM 105-positive membrane folds that extended along the length of the bile canalicular border. In sections from livers in which calcium-dependent adhesive contacts had been disrupted by treatment with ethylenediamine tetraacetate, intact bile canaliculi were found that remained attached only by these border folds. The implications of these results are discussed with regard to a possible role for cell-CAM 105 in bile canalicular formation.

Detection of cell-CAM 105 in the pericanalicular domain of the rat hepatocyte plasma membrane

Cell-CAM 105 has been identified as a cell adhesion molecule based on the ability of anti-cell-CAM 105 monospecific Fab fragments to inhibit the reaggregation of rat hepatocytes. Because of its adhesive properties, it was expected that cell-CAM 105 would be present on the lateral cell surface where adhesive interactions predominate. Paradoxically, however, immunofluorescence analysis of frozen sections of rat liver using specific monoclonal antibodies indicated that cell-CAM 105 was present exclusively in the bile canalicular domain of the rat hepatocyte where there is no intercellular adhesion. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase labeling and electron microscopy were used to examine intact and mechanically dissociated liver tissue. Results showed that when accessibility was provided by mechanical dissociation of perfusion fixed liver tissue, cell-CAM 105 could be detected in the pericanalicular region of lateral membranes. In contrast, when hepatocytes were labeled after incubation in vitro under conditions used during adhesion assays to induce reaggregation, cell-CAM 105 rapidly redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures further revealed that cell-CAM 105 and two other bile canalicular proteins relocalized to discrete domains reminiscent of bile canaliculi, whereas cell-CAM 105 was also present in areas of intercellular contact. Serial section electron microscopy analysis of well-defined, cross-sectional profiles of bile canaliculi suggested the presence of cell-CAM 105—positive membrane folds that extended along the length of the bile canalicular border. In sections from livers in which calcium-dependent adhesive contacts had been disrupted by treatment with ethylenediamine tetraacetate, intact bile canaliculi were found that remained attached only by these border folds. The implications of these results are discussed with regard to a possible role for cell-CAM 105 in bile canalicular formation. (HEPATOLOGY 1991; 13:47–56).

Effect of cadmium on F-actin and microtubules of Madin-Darby Canine Kidney cells

The effect of cadmium on F-actin and microtubules of Madin-Darby Canine Kidney cells was studied by cytochemical methods. A 6-hr exposure to cadmium (10 μm) in a buffered salt solution resulted in the breakdown of actin filaments, particularly those associated with both the stress fibers and the lateral membranes in areas of intercellular contact. Microtubules were not dramatically altered during this exposure period and cell viability, determined by trypan blue exclusion, was similar to controls. The effect of cadmium on actin was reversible if the cells were returned to culture medium. The results indicate that one possible mechanism of cadmium toxicity is via an effect on the organization of actin filaments.

Temporally specific involvement of cell surface β-1,4 galactosyltransferase during mouse embryo morula compaction

Cell surface β-1,4 galactosyltransferase (GalTase) is shown to mediate intercellular adhesions between embryonal carcinoma (EC) cells and specifically during late morula compaction in the preimplantation mouse embryo. Monospecific anti-GalTase IgG raised against affinity-purified bovine β-1,4 GaITase recognizes F9 EC cell GalTase as judged by immunoprecipitation and inhibition of GalTase activity, as well as by immunoprecipitation of a single 52 kd metabolically labeled membrane protein. Anti-GaITase IgG inhibits cell adhesions between EC cells, dissociates compacted mouse morulae, and inhibits blastocyst formation. Anti-GaITase IgG specifically inhibits cell adhesions during late morula compaction, coincident with a peak of surface GaITase activity as determined by direct enzyme assay. On EC cells, GaITase activity can be proteolytically released from intact cells, and is localized by indirect immunofluorescence to areas of intercellular contact, consistent with its proposed role in cell adhesion. β-1,4 GalTase is the first cell adhesion molecule identified that participates during late morula compaction, subsequent to uvomorulin function.

Localization of nonerythroid spectrin and actin in mouse oocytes and preimplantation embryos

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembraneous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembraneous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The sub-membraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells. Cytoskeletal components are probably involved in the formation of cell contacts and regulation of contact areas between blastomeres, as well as the control of cell movement. Thus, nonerythroid spectrin and actin may play important roles in the regulation of cleavage-stage development and blastocyst transformation.

Amphibian pronephric duct morphogenesis: segregation, cell rearrangement and directed migration of the Ambystoma duct rudiment

ABSTRACT The axolotl pronephric duct rudiment is readily accessible to both SEM observation and surgical manipulation. The rudiment segregates from the dorsal part of the lateral mesoderm and then extends caudally along the ventrolateral border of the segmenting somites, eventually contacting the cloacal wall. The marked thinning of the rudiment which accompanies this migration is paralleled by a corresponding reduction in cell number across the duct’s diameter and by caudad translocation and elongation of vital dye marks applied to the duct mesoderm. Duct extension thus involves appreciable cell rearrangement. The morphology of duct mesoderm and its substratum (somite and lateral mesoderm) suggests that active locomotion of cells near its tip marshals the duct’s caudad elongation. Filopodia and small focal areas of intercellular contact may mediate the adhesions between duct cells which must be broken and reformed as the cells rearrange.

PCC4azal teratocarcinoma stem cell differentiation in culture: II. Morphological characterization

The ultrastructural morphology of the PCC4azal embryonal carcinoma cells and their differentiated counterparts, endoderm-like cells and giant cells, was characterized and compared with that of the cells of embryoid bodies. The ultrastructure of the PCC4azal embryonal carcinoma cells is similar to that of the embryonal carcinoma cells of the embryoid body. These cells are small, with a large nucleus and relatively few cytoplasmic organelles. Gap junctions and modified adherens junctions are formed at some areas of intercellular contact between the embryonal carcinoma cells. The differentiated PCC4azal endoderm-like cells have a more developed cytoplasm, containing an extensive endoplasmic reticulum with large Golgi regions. Most striking is the de novo appearance of epithelial-like junctional complexes which join the apical borders between the endoderm-like cells, thus polarizing the cell monolayer. The zonula occludens junctions of the junctional complex are extensive, consisting of six or more strands of tight junctional ridges. Terminal webs are present in the apical regions that are inserted into the zonula adherens region of the junctional complex. Gap junctions continue to join neighboring cells, and some gap junctions are intercalated within tight junctional ridges. The ultrastructure of the differentiated endodermal cells of the embryoid bodies is very similar to that of the PCC4azal endoderm-like cells. The embryoid body endodermal cells form similar junctional complexes which also contain continuous belts of tight junctions that are intercalated with gap junctions. As the PCC4azal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of 10-nm filaments, microtubules, and 7-nm microfilaments. The junctional complexes that were present during the endodermal stage are partially disassembled as the giant cells migrate apart. Thus, the differentiation process in this system is characterized by significant and distinctive morphological changes.