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.

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

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.

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 Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

1990 ◽  
Vol 111 (5) ◽  
pp. 2077-2088 ◽  
Author(s):  
L S Musil ◽  
B A Cunningham ◽  
G M Edelman ◽  
D A Goodenough
Keyword(s):  
Cell Adhesion ◽  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
Gap Junctional Communication ◽  
Competent Cells ◽  
Deficient Cell ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

scholarly journals High-throughput measurement of gap junctional intercellular communication

2014 ◽  
Vol 306 (12) ◽  
pp. H1708-H1713 ◽  
Author(s):  
Jun Liu ◽  
Vinayakumar Siragam ◽  
Jun Chen ◽  
Michael D. Fridman ◽  
Robert M. Hamilton ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
High Throughput ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Cell Injection ◽  
Operation Speed ◽  
Gap Junctional

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Gap junctions and tumour progression

2002 ◽  
Vol 80 (2) ◽  
pp. 136-141 ◽  
Author(s):  
Christian CG Naus
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Tumour Progression ◽  
Second Messengers ◽  
Regulation Of Gene Expression ◽  
Growth Control ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junction Channels ◽  
Junctional Coupling ◽  
Gap Junctional

Gap junctional intercellular communication has been implicated in growth control and differentiation. The mechanisms by which connexins, the gap junction proteins, act as tumor suppressors are unclear. In this review, several different mechanisms are considered. Since transformation results in a loss of the differentiated state, one mechanism by which gap junctions may control tumour progression is to promote or enhance differentiation. Processes of differentiation and growth control are mediated at the genetic level. Thus, an alternative or complimentary mechanism of tumour suppression could involve the regulation of gene expression by connexins and gap junctional coupling. Finally, gap junction channels form a conduit between cells for the exchange of ions, second messengers, and small metabolites. It is clear that the sharing of these molecules can be rather selective and may be involved in growth control processes. In this review, examples will be discussed that provide evidence for each of these mechanisms. Taken together, these findings point to a variety of mechanims by which connexins and the gap junction channels that they form may control tumour progression.Key words: gap junctions, connexin, cancer.

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.

scholarly journals Gap Junctions Regulate Extracellular Signal-regulated Kinase Signaling to Affect Gene Transcription

2005 ◽  
Vol 16 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Joseph P. Stains ◽  
Roberto Civitelli
Keyword(s):  
Protein Kinase ◽  
Gap Junctions ◽  
Gap Junction ◽  
Gene Transcription ◽  
Electrical Coupling ◽  
Mitogen Activated Protein Kinase ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Extracellular Signal ◽  
Gap Junctional

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Iα1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.

scholarly journals Cardiac gap junction configuration after an uncoupling treatment as a function of time.

1979 ◽  
Vol 82 (1) ◽  
pp. 66-75 ◽  
Author(s):  
K M Baldwin
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Homogeneous Structure ◽  
Electrical Uncoupling ◽  
Gap Junctional ◽  
Injury Control ◽  
Junction Structure ◽  
Rabbit Atrium

Rabbit ventricle either was fixed in glutaraldehyde without injury (control) or was injured before fixation, presumably causing electrical uncoupling of the gap junctions. All tissue was then processed for freeze-fracture. Replicas of control gap junctions exhibited irregular packing of the P-face particles and E-face pits. Average center-to-center spacing of the particles was 10.5 nm. Tissue fixed 1-5 min after injury showed clumping of gap junctional particles and pits. Within the clumps, the particles and pits were hexagonally packed and the center-to-center spacing of the particles averaged 9.5 nm. In tissue fixed 15-30 min after injury, the clumps of gap junctional particles had coalesced into a homogeneous structure in most junctions. The packing of the particles and pits was hexagonal and the spacing of the particles averaged 9.5 nm. A few pieces of rabbit atrium were frozen without prior fixation or cryoprotection to try to assess the effect of glutarldehyde fixation on gap junction structure. In this tissue the gap junctional particles were irregularly packed and their spacing averaged 10.0 nm.

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