scholarly journals Hormonal regulation of gap junction differentiation.

1976 ◽  
Vol 69 (3) ◽  
pp. 669-685 ◽  
Author(s):  
R S Decker
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Hormonal Regulation ◽  
Large Particle ◽  
Rana Pipiens ◽  
Hormone Treatment ◽  
Junction Area ◽  
Plaque Area ◽  
New Protein

Thin-section, tracer, and freeze-cleave experiments on hypophysectomized Rana pipiens larvae reveal that gap junctions form between differentiating ependymoglial cells in response to thyroid hormone. These junctions assemble in large particle-free areas of the plasma membrane known as formation plaques. Between 20 and 40 h after hormone application, formation plaque area increases approximately 26-fold while gap junction area rises about 20-fold. The differentiation of these junctions requires the synthesis of new protein and probably RNA as well. On the basis of inhibitor experiments, it can be reported that formation plaques develop at about 16-20 h after hormone treatment and stages in the construction of gap junctions appear 4-8 h later. These studies suggest that gap junction subunits are synthesized and inserted into formation plaque membrane during the differentiation of the anuran ependymoglial cells.

scholarly journals Connexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization

2017 ◽  
Author(s):  
Rachael M. Kells-Andrews ◽  
Rachel A. Margraf ◽  
Charles G. Fisher ◽  
Matthias M. Falk
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Room Temperature ◽  
Cell Communication ◽  
Mapk Phosphorylation ◽  
Annular Gap ◽  
Summary Statement ◽  
Triton X

ABSTRACTGap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cell-to-cell communication by forming channels that connect the cytosols of adjacent cells. Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Interestingly, ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination may be triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination may be induced by Cx phosphorylation.Summary StatementHere we show that connexin 43 in gap junctions becomes K63-poly ubiquitinated on lysines 264 and 303 and its requirement for gap junction endocytosis. These novel findings significantly contribute to our understanding of GJ turnover and patho-/physiology.Abbreviations usedAGJannular gap junctionAMSHassociated molecule with the SH3 domain of STAMCMEclathrin-mediated endocytosisCxConnexinCx43Connexin 43DUBdeubiquitinaseGJgap junctionMonoUbmonoubiquitinNedd4-1neural precursor cell expressed developmentally down-regulated protein 4-1PMplasma membranePolyUbpolyubiquitinTPA12-O-Tetradecanoylphorbol 13-AcetateTX-100Triton X-100RTroom temperatureUbubiquitin

Changes of gap junctions in myometrium of guinea pig at parturition and abortion

1982 ◽  
Vol 60 (3) ◽  
pp. 335-341 ◽  
Author(s):  
R. E. Garfield ◽  
E. E. Daniel ◽  
M. Dukes ◽  
J. D. Fitzgerald
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Gap Junctions ◽  
Gap Junction ◽  
Electron Micrographs ◽  
Guinea Pigs ◽  
Muscle Cells ◽  
Similar Increase ◽  
Junction Area

Myometrial tissues from guinea pigs were quantitatively examined for gap junctions in electron micrographs. Small numbers of gap junctions were present between smooth muscle cells in myometria of pregnant guinea pigs at days 50 and 65 of gestation. The junctions increased in number and size at parturition on day 69 and decreased again to control levels 24 h after parturition. A similar increase in junctions occurred when abortion was induced by 16,16-dimethylprostaglandin E2 (PGE2) on day 65. There were no consistent or significant differences in numbers of gap junctions from myometrium taken over sites of placental attachment and from other sites. These results together with previous studies suggest that an increase in myometrial gap junction area is associated with and may be essential for parturition in guinea pigs, but the control of their development may differ from that in other mammals.

scholarly journals Trafficking, Assembly, and Function of a Connexin43-Green Fluorescent Protein Chimera in Live Mammalian Cells

1999 ◽  
Vol 10 (6) ◽  
pp. 2033-2050 ◽  
Author(s):  
Karen Jordan ◽  
Joell L. Solan ◽  
Michel Dominguez ◽  
Michael Sia ◽  
Art Hand ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Dye Transfer ◽  
Green Fluorescent

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin–Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 μm and 0.5–1.5 μm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

scholarly journals Preparation of a gap junction fraction from uteri of pregnant rats: the 28-kD polypeptides of uterus, liver, and heart gap junctions are homologous.

1985 ◽  
Vol 101 (4) ◽  
pp. 1363-1370 ◽  
Author(s):  
A S Zervos ◽  
J Hope ◽  
W H Evans
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Apparent Molecular Weight ◽  
Antigenic Determinants ◽  
Pregnant Rats ◽  
Thin Sections ◽  
Amino Terminal ◽  
Junction Protein ◽  
Gap Junction Protein

A procedure for the preparation of a gap junction fraction from the uteri of pregnant rats is described. The uterine gap junctions, when examined by electron microscopy of thin sections and in negatively stained preparations, were similar to gap junctions isolated from heart and liver. Major proteins of similar apparent molecular weight (Mr 28,000) were found in gap junction fractions isolated from the uterus, heart, and liver, and were shown to have highly homologous structures by two-dimensional mapping of their tryptic peptides. An Mr 10,000 polypeptide, previously deduced to be a proteolytic product of the Mr 28,000 polypeptide of rat liver (Nicholson, B. J., L. J. Takemoto, M. W. Hunkapiller, L. E. Hood, and J.-P. Revel, 1983, Cell, 32:967-978), was also studied and shown by chymotryptic mapping to be homologous in the uterine, heart, and liver gap junction fractions. An antibody raised in rabbits to a synthetic peptide corresponding to an amino-terminal sequence of the liver gap junction protein recognized Mr 28,000 proteins in the three tissues studied, showing that the proteins shared common antigenic determinants. These results indicate that gap junctions are biochemically conserved plasma membrane specializations. The view that gap junctions are tissue-specific plasma membrane organelles based on previous comparisons of Mr 26,000-30,000 polypeptides is not sustained by the present results.

Connexin 43 ubiquitination determines the fate of gap junctions: restrict to survive

2015 ◽  
Vol 43 (3) ◽  
pp. 471-475 ◽  
Author(s):  
Teresa M. Ribeiro-Rodrigues ◽  
Steve Catarino ◽  
Maria J. Pinho ◽  
Paulo Pereira ◽  
Henrique Girao
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Transmembrane Proteins ◽  
Post Translational Modifications ◽  
Gap Junction Intercellular Communication

Connexins (Cxs) are transmembrane proteins that form channels which allow direct intercellular communication (IC) between neighbouring cells via gap junctions. Mechanisms that modulate the amount of channels at the plasma membrane have emerged as important regulators of IC and their de-regulation has been associated with various diseases. Although Cx-mediated IC can be modulated by different mechanisms, ubiquitination has been described as one of the major post-translational modifications involved in Cx regulation and consequently IC. In this review, we focus on the role of ubiquitin and its effect on gap junction intercellular communication.

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 (&lt; 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.

scholarly journals Opaque deposits on gap junction membranes after glutaraldehyde-calcium fixation.

1975 ◽  
Vol 67 (3) ◽  
pp. 801-813 ◽  
Author(s):  
W J Larsen
Keyword(s):  
Heavy Metal ◽  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Room Temperature ◽  
Asymmetric Distribution ◽  
Hybrid Cells ◽  
Fixed Membrane ◽  
Gap Junctional ◽  
Paired Cell

When cloned hybrid cells (A/Bm-5) were grown to confluence and fixed in glutaraldehyde-calcium, electron-opaque deposits were observed on the cytoplasmic faces of plasma membrane. Deposits were most abundant at gap junctions. Deposits were often precisely paired, cell-to-cell, across the gap junctional membranes, and these paired deposits were frequently equivalent in size. This relationship was most often observed on long profiles of gap junctions, in contrast to the asymmetric distribution of larger deposits commonly found on short junctional profiles. Deposits were present with or without heavy metal staining but did not appear when calcium was omitted from the fixative. Fixation at room temperature yielded more and larger deposits than fixation at 0 degrees C. The significance of these observations is discussed with regard to the possible binding of calcium at fixed membrane sites or the precipitation of calcium by anions produced by enzymes located at the gap junction.

Effects of tamoxifen citrate and cycloheximide on estradiol induction of rat myometrial gap junctions

1986 ◽  
Vol 64 (6) ◽  
pp. 703-706 ◽  
Author(s):  
L. W. MacKenzie ◽  
R. E. Garfield
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Hormone Treatment ◽  
Control Groups ◽  
Estradiol Treatment ◽  
Myometrial Cells ◽  
Tamoxifen Citrate ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Junction Proteins

Longitudinal muscle of myometrial tissues from immature rats were examined by quantitative thin section electron microscopy for the presence of gap junctions after treatment with estradiol with and without tamoxifen, and cycloheximide for 1–6 days. Gap junctions were present between myometrial cells on days 4, 5, and 6 after treatment with estradiol (500 μg/day). Tamoxifen administered concomitantly with estradiol over the 6-day period completely prevented induction of the junctions. Gap junctions were not detected in the myometrium after treatment with tamoxifen alone. Administration of cycloheximide together with estradiol on day 0 of the 6-day period had no effect on gap junction frequency but resulted in a reduction in gap junction size in the myometrium after continued treatment with the hormone. Treatment with cycloheximide on day 1, however, significantly suppressed the effect of further estradiol treatment on the induction of gap junctions in the myometrium. Junctions were not visible in the tissues from animals treated with cycloheximide alone or in the control groups treated with sesame oil. These results indicate that estradiol influences the presence of gap junctions in the myometrium by regulating the synthesis of gap junction proteins through the steroid receptor mechanism.

scholarly journals Cytosolic Stress Reduces Degradation of Connexin43 Internalized from the Cell Surface and Enhances Gap Junction Formation and Function

2005 ◽  
Vol 16 (11) ◽  
pp. 5247-5257 ◽  
Author(s):  
Judy K. VanSlyke ◽  
Linda S. Musil
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junctions ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Proteasome Inhibitors ◽  
Cell Surface Biotinylation ◽  
Normal Rat ◽  
And Function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.

scholarly journals Preparation of hepatic gap (communicating) junctions. Identification of the constituent polypeptide subunits

1977 ◽  
Vol 168 (3) ◽  
pp. 475-481 ◽  
Author(s):  
Janetta G. Culvenor ◽  
W. Howard Evans
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Density Perturbation ◽  
Morphological Properties ◽  
Communicating Junctions ◽  
Transmembrane Channels ◽  
A Minor

1. Gap (communicating) junctions are plasma-membrane specializations of characteristic morphology that form transmembrane channels allowing direct communication between cells. Their preparation is described starting from mouse liver plasma membranes and the constituent polypeptides are deduced. 2. Gap junctions co-purify with collagen fibres when the plasma-membrane residues insoluble in N-dodecyl sarcosinate are fractionated on sucrose gradients. Sucrose-density perturbation by relipidation of isolated gap junctions or the use of urea to remove non-junctional membranes both failed to diminish the collagen content of fractions. 3. Removal of collagen by treatment with purified collagenase preparations yielded morphologically satisfactory gap-junction fractions. Analysis by polyacrylamide-gel electrophoresis of the polypeptides present in gap junctions prepared by procedures omitting or using collagenases indicated two non-glycosylated polypeptides, a major component of apparent mol.wt. 38000 and a minor 40000-mol.wt. component. These two polypeptides were also present in plasma membranes and the intermediate fractions. 4. Proteolysis of the gap-junction polypeptides yielding components of mol.wt. 34000, 25000 and below 20000 occurred when iodinated gap junctions were subject to prolonged collagenase treatment, thus explaining the variable polypeptide composition of gap junctions reported by others. 5. The morphological properties of the isolated gap junctions prepared by the various procedures are described.

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