Synthesis and assembly of human beta 1 gap junctions in BHK cells by DNA transfection with the human beta 1 cDNA

1995 ◽  
Vol 108 (12) ◽  
pp. 3725-3734 ◽  
Author(s):  
N.M. Kumar ◽  
D.S. Friend ◽  
N.B. Gilula
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Growth Control ◽  
Double Membrane ◽  
Correct Orientation ◽  
Junctional Communication ◽  
Junction Protein ◽  
Particle Aggregates ◽  
Metallothionein Promoter

Gap junctional communication is important in many physiological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human beta 1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional beta 1 connexin was detected only in cells transfected with beta 1 connexin cDNA in the correct orientation (beta 1-BHK). Analysis of the cells by light microscopic immunocytochemistry indicated that beta 1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomerization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.

scholarly journals Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly.

1994 ◽  
Vol 127 (6) ◽  
pp. 1895-1905 ◽  
Author(s):  
P D Lampe
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Freeze Fracture ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Junctional Communication ◽  
Junction Protein

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

scholarly journals Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763 ◽  
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

scholarly journals Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

1990 ◽  
Vol 1 (13) ◽  
pp. 989-1002 ◽  
Author(s):  
K I Swenson ◽  
H Piwnica-Worms ◽  
H McNamee ◽  
D L Paul
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Expression System ◽  
Functional Expression ◽  
Gap Junction Communication ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junction Protein

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.

scholarly journals Facets of Communication: Gap Junction Ultrastructure and Function in Cancer Stem Cells and Tumor Cells

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 288 ◽  
Author(s):  
Anja Beckmann ◽  
Nadine Hainz ◽  
Thomas Tschernig ◽  
Carola Meier
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Gap Junctions ◽  
Gap Junction ◽  
Cancer Cells ◽  
Freeze Fracture ◽  
Plaque Morphology ◽  
Junctional Communication ◽  
Gap Junction Proteins ◽  
Junction Proteins

Gap junction proteins are expressed in cancer stem cells and non-stem cancer cells of many tumors. As the morphology and assembly of gap junction channels are crucial for their function in intercellular communication, one focus of our review is to outline the data on gap junction plaque morphology available for cancer cells. Electron microscopic studies and freeze-fracture analyses on gap junction ultrastructure in cancer are summarized. As the presence of gap junctions is relevant in solid tumors, we exemplarily outline their role in glioblastomas and in breast cancer. These were also shown to contain cancer stem cells, which are an essential cause of tumor onset and of tumor transmission into metastases. For these processes, gap junctional communication was shown to be important and thus we summarize, how the expression of gap junction proteins and the resulting communication between cancer stem cells and their surrounding cells contributes to the dissemination of cancer stem cells via blood or lymphatic vessels. Based on their importance for tumors and metastases, future cancer-specific therapies are expected to address gap junction proteins. In turn, gap junctions also seem to contribute to the unattainability of cancer stem cells by certain treatments and might thus contribute to therapeutic resistance.

The relationship of gap junctions and compaction in the preimplantation mouse embryo

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 113-118
Author(s):  
David L. Becker ◽  
Catherine Leclerc-David ◽  
Anne Warner
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gap Junctional Communication ◽  
Stage Of Development ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional

In the mouse embryo, gap junctions first appear at the 8-cell stage as compaction is about to take place. Compaction of the embryo is important for the differentiation of the first two cell types; the inner cell mass and the trophectoderm. Our studies examine the contribution of gap junctional communication at this stage of development We have characterised the normal sequence of appearance of gap junction protein and its distribution. The extent of communication as shown by the passage of dye between cells has been recorded in both normal embryos and embryos treated with drugs that influence gap junctional communication. Comparisons have been made with embryos that express a lethal gap junction defect and attempts were made to rescue such embryos by increasing their gap junction communication.

Freeze-fracture studies of frog atrial fibres

1976 ◽  
Vol 22 (2) ◽  
pp. 427-434
Author(s):  
F. Mazet ◽  
J. Cartaud
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Outer Membrane ◽  
Electrical Coupling ◽  
Freeze Fracture ◽  
Present Knowledge ◽  
Inner Membrane ◽  
Freeze Fracturing ◽  
Morphological Variant ◽  
Characteristic Features

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the “gap junction” or “nexus”. The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.

scholarly journals Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.

1991 ◽  
Vol 115 (5) ◽  
pp. 1357-1374 ◽  
Author(s):  
L S Musil ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Surface Protein ◽  
Immunohistochemical Localization ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional ◽  
Triton X

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

scholarly journals Gap junction structures after experimental alteration of junctional channel conductance.

1985 ◽  
Vol 101 (5) ◽  
pp. 1741-1748 ◽  
Author(s):  
T M Miller ◽  
D A Goodenough
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Electron Micrographs ◽  
Time Course ◽  
Structural Changes ◽  
Lucifer Yellow ◽  
Freeze Fracture ◽  
Channel Conductance ◽  
Experimental Alteration ◽  
Quick Freezing

Gap junctions are known to present a variety of different morphologies in electron micrographs and x-ray diffraction patterns. This variation in structure is not only seen between gap junctions in different tissues and organisms, but also within a given tissue. In an attempt to understand the physiological meaning of some aspects of this variability, gap junction structure was studied following experimental manipulation of junctional channel conductance. Both physiological and morphological experiments were performed on gap junctions joining stage 20-23 chick embryo lens epithelial cells. Channel conductance was experimentally altered by using five different experimental manipulations, and assayed for conductance changes by observing the intercellular diffusion of Lucifer Yellow CH. All structural measurements were made on electron micrographs of freeze-fracture replicas after quick-freezing of specimens from the living state; for comparison, aldehyde-fixed specimens were measured as well. Analysis of the data generated as a result of this study revealed no common statistically significant changes in the intrajunctional packing of connexons in the membrane plane as a result of experimental alteration of junctional channel conductance, although some of the experimental manipulations used to alter junctional conductance did produce significant structural changes. Aldehyde fixation caused a dramatic condensation of connexon packing, a result not observed with any of the five experimental uncoupling conditions over the 40-min time course of the experiments.

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 509-522
Author(s):  
R. Minkoff ◽  
S.B. Parker ◽  
E.L. Hertzberg
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Uniform Distribution ◽  
Rat Liver ◽  
Connexin 43 ◽  
Cell Communication ◽  
Exterior Surface ◽  
Junction Protein ◽  
Primary Palate ◽  
Gap Junction Protein

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 × 10(3) Mr, connexin 32) and heart (43 × 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 × 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the ‘heart’ 43 × 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

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