Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Intercellular Junctions”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMed2002

Correlation of a dynamic model for immunological synapse formation with effector functions: two pathways to synapse formation.

During antigen recognition by T cells different receptors and ligands form a pattern in the intercellular junction called the immunological synapse, which might be involved in T-cell activation. Recently, a synapse assembly model has been proposed, which enables the calculation of the propensity for synapse assembly driven by membrane-constrained protein binding interactions. We bring together model predictions of mature synapse assembly with data on the dependence of T-cell responses on T-cell receptor (TCR)-MHC-peptide (pMHC) binding kinetics. Predictions of mature synapse assembly, based on TCR-pMHC binding kinetics, correlate well with observed cytokine responses by T cells bearing the relevant TCR but not with cytotoxic T lymphocyte-mediated killing. We discuss the suggested different role for the synapse in pre- and post-nuclear activation events in T cells. The view of immunological synapse assembly given here emphasizes the importance of both the on and off rates for the TCR-pMHC interaction and in this context recent data on a positive role for analogs of self-peptides in synapse assembly is considered.

Animals↗
PubMed1980

The development of the sinusoids of fetal rat liver: morphology of endothelial cells, Kupffer cells, and the transmural migration of blood cells into the sinusoids.

The fine structural development of rat fetal liver sinusoids from 10 to 22 days gestation was studied. Colloidal carbon (Pelikan ink) was injected into 14-22 day gestation fetuses via the umbilical vein to assess the continuity of the sinusoidal lining and the phagocytic ability of the developing lining cells. Endothelial cells, devoid of an underlying basal lamina, form the bulk of the vascular lining at all gestational ages. These cells possess typical intercellular junctions and fenestrae with diaphragms before 17 days gestation. Transendothelial open fenestrations, typical of the adult liver, appear around 17 days gestation, increasing in number for the remainder of gestation. Although fenestrae possessing diaphragms are permeable to carbon before 16 days gestation, open fenestrations, first seen at 17 days gestation, allowed large amounts of carbon to reach the extravascular space. Endocytosis of carbon by endothelial cells was accomplished exclusively by large bristle-coated vesicles. Endothelial cells were also seen to be involved in transmural diapedesis of newly formed erythrocytes and megakaryocyte processes from the extravascular space by forming a temporary migration pore allowing these cells and processes to enter the circulation. At the end of gestation, blood-forming activity had nearly ceased, and only the space of Dissé separated the lining cells from the parenchymal cells. Kupffer cells were easily identified as early as 13 days gestation by their content of phagosomes and engulfed erythrocytes. The Kupffer cells are much more avid in the phagocytosis of carbon than are endothelial cells. Toward the end of gestation, some Kupffer cells develop a homogeneous "sticky coat" to carbon.

Animals↗
PubMed1988

The polarized distribution of an apical cell surface glycoprotein is maintained by interactions with the cytoskeleton of Madin-Darby canine kidney cells.

A monoclonal antibody made against a 135-kD glycoprotein (gp135) on the plasma membrane of Madin-Darby canine kidney (MDCK) cells was used to study the development and maintenance of epithelial cell surface polarity. Immunofluorescence microscopy and immunogold electron microscopy of confluent monolayers demonstrated that gp135 had a polarized cell surface distribution and was only localized on the apical surface. The role of membrane contacts in establishing gp135 polarity was determined by plating cells in low Ca++-medium to prevent the formation of intercellular junctions. Quantitative immunogold electron microscopy demonstrated that gp135 had a polarized distribution on cells lacking membrane contacts and was observed on the apical surface at a density 24 times that of the basal membrane contacting the substratum. The possibility that gp135 was associated with components of the apical cytoskeleton was investigated using cytoskeleton-disrupting drugs. Incubation in cytochalasin D produced a clustering of both actin and gp135, and double-label fluorescence microscopy demonstrated that these proteins were colocalized. Experiments using nocodazole had no effect, suggesting that gp135 could be interacting with actin microfilaments, but not microtubules. Treatment with Triton X-100 extracted approximately 50% of the gp135 and immunofluorescence microscopy indicated that the gp135 which remained associated with the detergent-insoluble cytoskeleton had a distribution identical to that of control cells. Experiments demonstrating that gp23, a nonpolarized glycoprotein, was preferentially extracted from the apical membrane suggested that the improperly sorted apical gp23 did not interact with the cytoskeleton. These results provided evidence that the polarized cell surface distribution of gp135 was maintained through its interaction with actin in the apical cytoskeleton.

Actins↗
PubMedDate not supplied

Electron microscopic observations in a case of long-standing profound sensorineural deafness.

The ultrastructural pathology in an ear with long-standing profound deafness is presented. Endolymphatic hydrops was present, although there had been no vestibular symptoms. Although hair cells were absent, the intercellular junctional complex at the endolymphatic surface of the organ of Corti was maintained, suggesting "healing" by migration or proliferation of supporting cells to fill the empty spaces. Severe atrophy of the stria vascularis was present, and crystalline deposits were found within the strial remnant. An encapsulated tectorial membrane was interpreted in light of ultrastructural findings as either herniation of the fibrillar connective tissue of the spiral limbus into the scala media or alternatively demonstrating abnormal continuity between the connective tissue of the spiral limbus and the remnant of tectorial membrane. Neural degeneration was severe in all cochlear turns. However, more nerve fibers were seen in the proximal portion of Rosenthal's canal than at the habenula perforata or in the organ of Corti, and central processes from the ganglion area were more numerous than peripheral processes. The ganglion cell population was reduced to approximately 10% of normal, and only unmyelinated cell bodies were found. Few recognizable neural fibers were seen within the organ of Corti, although bizarre collections of apparent cellular debris and cell processes containing many mitochondria or vesicles were seen in regions normally containing neural fibers.

Atrophy↗
PubMed1991

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes.

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.

Basement Membrane↗
PubMed2001

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells.

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.

Animals↗
PubMed1985

Anatomy and physiology of the pleural space.

The early development of a coelom, a primitive body cavity, with stretchable mesothelial cells endows the subsequently developed internal organs a great flexibility to expand, retract, and deform. In the pleural cavity, in addition, the lung is maintained in an inflated state by the mechanical coupling between the chest wall and the lung; the mesothelial cells with bush-like elongated microvilli enmesh hyaluronic acid-rich lubricants; both minimize the work of breathing. Normal mesothelial cells are fragile in the air, and their intercellular junctions are narrow but labile. The activated mesothelial cells, on the other hand, are resilient and rich in organelles and enzymes to partake in functions such as fibrinolysis, in order to maintain the patency of the pleural cavity. Fluid and electrolytes permeate freely between normal mesothelial cells; the fluid moves in and out of the pleural cavity following Starling's law, with the endothelium as the main barrier. Transudates in the pleural cavity are formed from alterations of the hydrostatic-osmotic pressure relationship, which normally keeps the pleural cavity dry. More than one factor is usually involved in the formation of exudates. Proteins, particles, and cells in the pleural exudates are removed mainly from the preformed stomas and the lymphatic lacuna present in the lower mediastinum and also in the subcostal region and portions of the diaphragm. This removal of the pleural fluid and particles by the lymphatic route is enhanced by the respiratory movements and is partly responsible for the topographic differences of the pleural pressure. Kampmeier's foci found in the location of the stomas are conglomerates of activated mesothelial and lymphoreticular cells with central capillary and lymphatic vessels. They act like tonsils to impede direct entry of undesirable materials from the pleural cavity into the chest wall and mediastinum.

Animals↗
PubMed2005

ZO-1: lamellipodial localization in a corneal fibroblast wound model.

PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.

Animals↗
PubMed1999

Characterization of the 4C8 antigen involved in transendothelial migration of CD26(hi) T cells after tight adhesion to human umbilical vein endothelial cell monolayers.

In extravasation of T cells, little is known about the mechanisms of transendothelial migration subsequent to the T cells' tight adhesion to endothelium. To investigate these mechanisms, we developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not adhesion in a culture system in which high CD26-expressing (CD26(hi)) T cells preferentially migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. Anti-4C8 reacted with all CD3(+) T cells and monocytes but not neutrophils or HUVECs. The structure defined by this antibody was an 80-kD molecule. The mAb at 1 mug/ml inhibited 80-90% of migration of CD3(+) T cells through unstimulated and interferon gamma-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVECs in the presence of anti-4C8. Anti-4C8 exhibited agonistic effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 mug/ml. The predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26(hi). Second, solid-phase-immobilized anti-4C8 induced adhesion of T cells to the substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26(hi) T cells when added to T cells at a high dose of 10 mug/ml. Finally, both anti-4C8-induced chemokinetic migration and transendothelial migration were inhibited by pretreatment of T cells with pertussis toxin. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26(hi) cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be involved in preferential transmigration of CD26(hi) cells adherent to HUVECs.

Actins↗
PubMed1999

Effect of inflammatory stimuli on the silver staining pattern of the rat carotid endothelium.

Silver nitrate stains the intercellular junctions of the endothelium and other cytoplasmic or membrane components. Two protocols are described for the silver staining of rat carotid endothelium that exclude the use of pressurized fixatives and simplify the technique previously described for rat aorta. The entire surface of the carotid endothelium was examined and several parameters (stigmata, granularity, clustering of anionic sites, transversal lines, weakening of silver lines and leukocyte adhesion) were evaluated. We studied the pattern of silver staining in two situations: (1) endothelial activation and (2) neurogenic inflammation. Endothelial activation was produced by the intravenous administration of a proinflammatory albumin or polyinosinic acid. Both products cause a marked increase in leukocyte adhesion concomitant with a decrease in argyrophilia and a weakness or loss of silver lines. Neurogenic inflammation, which is mediated by substances released from sensory nerves, was induced by the intravenous administration of substance P or capsaicin. Both stimuli produced an increase in argyrophilia and weakness or loss of silver lines. Substance P caused a clustering of anionic sites, whereas this phenomenon was more discrete with capsaicin. Nearly 80% of all examined rats (controls and inflammatory stimuli treated) showed endothelial membrane disruptions formed by clusters of cells often in the shape of streaks aligned with the long axis of the vessel. The detection of these discontinuities is important, as loss of endothelial integrity is central in the initiation of pathological events.

Animals↗
PubMed1992

Cellular and molecular aspects of PECAM-1.

PECAM-1 is a surface membrane glycoprotein of 130,000 daltons found on platelets, endothelial cells, monocytes, neutrophils, and certain T-cell subsets. Except for bone marrow precursor cells, it does not seem to be found outside the vasculature. Previous studies have shown that PECAM-1 is a member of the immunoglobulin gene superfamily that localizes to the intercellular junction in cells, like endothelial cells, that grow in monolayers. We and other laboratories have been involved in studies designed to define the role of PECAM-1 in various cell types. Mammalian expression vectors have been constructed that express all, or selected domains, of the PECAM-1 protein. These vectors have been used to induce PECAM-1 expression in a variety of both monolayer and suspension cell types. Domain-specific anti-PECAM-1 monoclonal antibodies, synthetic peptides, and heparin were also used to define structurally and functionally important domains of this membrane glycoprotein. Protein synthesis, subcellular localization, and association with the cytoskeletons of various cells expressing PECAM-1 were examined. From these studies we have obtained evidence that: (1) PECAM-1 redistributes to the border of monolayer cells when they contact each other. (2) Border localization may result from homophilic interactions, i.e. PECAM-1 on one cell binds PECAM-1 on the adjacent cell. (3) Suspension cells such as L-cells or neutrophils may also interact via PECAM-1 mediated interactions, but an as yet unidentified ligand probably participates in a heterophilic, rather than homophilic, type of interaction. (4) Intracellular movement of PECAM-1 in both monolayer and suspension cell types requires active participation of the cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis.

This brief review details the structure, nature, and distribution of the fibronexus, and discusses its significance for myofibroblastic differentiation and tumor diagnosis. The fibronexus is a cell surface specialization consisting of intracellular actin filaments and extracellular fibronectin filaments associated with subplasmalemmal plaque material. The fibronexus represents an intercellular junction between myofibroblasts, but in particular is a device for providing contact between myofibroblasts and matrix that mediates continuity between intracellular contractile filaments and extracellular matrix proteins. Immunoelectron microscopy in particular has shown that the intracellular filaments contain actin. The extracellular filaments contain fibronectin and collectively form the fibronectin fibril. The plaque probably contains such proteins as vinculin, talin, alpha-actinin, and integrin. Under appropriate biologic development and fixation conditions, the fibronectin fibril of the fibronexus is characterized by and distinguished from lamina by enhanced density, a rigid appearance, failure to adhere closely to the contours of the cell surface (except focally near the plaque material), and a longitudinally filamentous substructure. Confirmation of the presence of a fibronectin fibril may be obtained by the finding of intense cell surface staining with an antifibronectin antibody. Problems in identifying the fibronexus may be encountered, however, due to poor development and fixation, in which case the filamentous substructure may be inapparent. The fibronexus is such a typical feature of and is often so conspicuous in myofibroblasts that it can be regarded as perhaps essential for the interpretation of myofibroblastic differentiation. Structures with a similar appearance have been documented in fundamentally nonmyofibroblastic cells; these include aortic and scleral spur smooth muscle cells and endothelium. Uncertainties remain in the protein composition of the fibronexus, the nature of its contact with the matrix, and its relationship to similar structures seen in nonmyofibroblastic cells. Immunoelectron microscopy provides a potential means of clarifying some of these questions.

Actins↗
PubMed1993

Ultrastructure of fiber cells and multilamellar inclusions in experimental diabetes.

PURPOSE: The goal of this ultrastructural study was to examine fiber cell shape and intercellular junctions during the early stages of fiber cell breakdown and edema in diabetic rabbit lenses. METHODS: Lens abnormalities were recorded with a slit lamp. Between 6-10 mo after drug treatment, diabetic lenses and untreated control lenses were freshly enucleated and sectioned with a vibrating knife microtome. The thick tissue sections were chemically fixed and processed for thin-section electron microscopy. RESULTS: Alloxan-induced diabetes in albino rabbits produced clinically apparent cataracts as soon as 1 mo after the animals became hyperglycemic. The cataracts displayed cortical fluid-filled vacuoles in the equatorial region and at the cortex-nucleus interface, white specks scattered throughout the cortex, and posterior subcapsular cataracts. Fiber cells just deeper than the large cortical vacuoles had oval or spindle-shaped cross sections. Multilamellar inclusions, not reported previously for diabetic lenses, were observed at or near the fiber cell interfaces and were composed of concentric or spiral rings of plasma membrane-bound cytoplasmic processes. Undulating membranes were present throughout most of the multilamellar inclusions. Transparent lenses from untreated controls did not have such multilamellar bodies or extensive membrane undulations in cells at the same distance from the lens surface. CONCLUSIONS: Fiber cells respond to the diabetic insult differently depending on their stage of differentiation and age. The observed changes are consistent with the hypothesis that hyperglycemia accelerates the formation of age-related changes in fiber cells.

Alloxan↗
PubMed2004

7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells.

BACKGROUND: Connexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols) affect gap junction intercellular communication (GJIC). RESULTS: Primary cultures of lens epithelial cells (LEC) were incubated with 7-ketocholesterol (7-Keto), 25-hydroxycholesterol (25-OH) or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake. CONCLUSIONS: Results demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication, that is most likely due to an increased stability of protein at the plasma membrane and to increased abundance of Cx43 assembled in gap junction plaques.

Journal Article↗
PubMed1984

Freeze-fracture of 2-cell mouse embryos. A new method for fracture of very small and scarce biological samples.

Membrane morphology and development of intercellular junctions in 2-cell mouse embryos was studied with freeze-fracture electron microscopy. In order to circumvent the dual problem of relative scarcity of samples and their very small size (about 50 micrometers per blastomere), both of which made conventional microtomy-induced freeze-cleavage impractical, a new protocol for handling 2-cell embryos was derived from freeze-fracture procedures established for monolayer cultures. Freshly-collected 2-cell embryos were placed within a 2 mm2 area on plastic Thermonox or Polystyrene coverslips which had been previously coated with 0.002% poly-l-lysine. The embryos were then fixed in 2.5% glutaraldehyde buffered to pH 7.3 with sodium cacodylate and containing 0.01% ruthenium red. The embryos were washed three times in buffer and then glycerinated. The next day, the 2 mm2 area of coverslip containing the embryos (75-100) was cut out with a fine scalpel, inverted and placed on a drop of Elvanol-glycerol mixture and frozen in a slurry of liquid nitrogen-cooled Freon-22. Fracturing was achieved in a Balzers BAF 301 Freeze-Etch Unit by positioning the microtome blade underneath a corner of the inverted coverslip and raising the knife until the coverslip 'popped off'. This procedure left the surface of the Elvanol droplet covered with fractured embryos. The exposed tissue was shadowed with platinum and coated with carbon. This protocol produces large expanses of blastomere plasma membrane as well as cross-fractures of entire blastomeres and 2-cell embryos. The replicas revealed blastomeres endowed with a rich assortment of pleomorphic microvilli.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia.

Ischemic epithelial cells are characterized by disruption of intercellular junctions and loss of apical-basolateral protein polarity, which are normally dependent on the integrity of the adherens junction (AJ). Biochemical analysis of both whole ischemic kidneys and ATP-depleted Madin-Darby canine kidney (MDCK) cells demonstrated a striking loss of E-cadherin (the transmembrane protein of the AJ) with the appearance and accumulation of an approximately 80-kDa fragment reactive with anti-E-cadherin antibodies on Western blots of ATP-depleted MDCK cells. This apparent ischemia-induced degradation of E-cadherin was not blocked by either inhibitors of the major proteolytic pathways (i.e., proteasome, lysosome, or calpain), or by chelation of intracellular calcium, suggesting the involvement of a protease capable of functioning at low ATP and low calcium levels. Immunocytochemistry revealed the movement of several proteins normally comprising the AJ, including E-cadherin and beta-catenin, away from lateral portions of the plasma membrane to intracellular sites. Moreover, rate-zonal centrifugation and immunoprecipitation with anti-E-cadherin and anti-beta-catenin antibodies indicated that ATP depletion disrupted normal E-cadherin-catenin interactions, resulting in the dissociation of alpha- and gamma-catenin from E-cadherin and beta-catenin-containing complexes. Because the generation and maintenance of polarized epithelial cells are dependent upon E-cadherin-mediated cell-cell adhesion and normal AJ function, we propose that the rapid degradation of E-cadherin and dissolution of the AJ is a key step in the development of the ischemic epithelial cell phenotype. Furthermore, we hypothesize that the reassembly of the AJ after ischemia/ATP depletion may require a novel bioassembly mechanism involving recombination of newly synthesized and sorted E-cadherin with preexisting pools of catenins that have (temporally) redistributed intracellularly.

Adenosine Triphosphate↗
PubMed1998

Molecular organization of the desmoglein-plakoglobin complex.

Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and alpha-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with alpha-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent co-immunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1-3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with alpha-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as alpha-catenin and desmosomal cadherins.

Alanine↗
PubMed1985

Gap junctions and direct intercellular communication between rat uterine smooth muscle cells.

We have tested the hypothesis that an increase in direct intercellular communication accompanies the development of gap junctions (GJs) between rat uterine smooth muscle cells at parturition. Intercellular communication in these tissues was studied by exposing one portion of small strips of myometrium to 2-[3H]deoxy-D-glucose (2-DG) and determining the longitudinal distribution of tracer after a 5-h period of diffusion. The distribution of 2-DG was greater in parturient compared with ante- and postpartum tissues. Similarly, the apparent diffusion coefficient of 2-DG was almost 10-fold greater in delivering tissues (1.86 X 10(-6) cm2/s) than before (0.199 X 10(-6) cm2/s) or after (0.296 X 10(-6) cm2/s) parturition. Control experiments indicated that the redistribution of 2-DG was dependent on the presence of GJs and was the result of intracellular and direct cell-to-cell diffusion. The appearance of GJs is the myometrium at term facilitates direct intercellular communication between uterine smooth muscle cells during labor. This improved communication may be responsible for synchronizing and coordinating electrical, metabolic, and contractile activity in the uterine wall and, hence, the effective expulsion of fetuses.

Animals↗