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

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.     

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

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.     

Innexins form two types of channels

Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non-junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.     

Structural,immunocytochemical and initial biochemical characterization of NAOH-extracted gap junctions from an insect,Heliothis virescens

Subcellular fractions enriched in gap junctions with an ultrastructure similar to those in intact insect tissue have been obtained by extracting crude membranes from the tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) with 2.5 mM NaOH. n-Octyl--d-glucopyranoside (OG) was used to further purify integral membrane proteins in the NaOH-extracted fractions. A polyclonal antibody (R16) is described that specifically labels nonextracted and NaOH-extracted gap junctions in cell fractions by electron microscope immunocytochemistry. R16 immunostaining of sectioned Heliothis testis at the light-microscope level yields a pattern of immunoreactivity consistent with the distribution of gap junctions in the tissue. R16 identifies a 40-kDa protein as a candidate gap junction protein on immunoblots of crude membrane, NaOH-extracted and NaOH/OG-extracted fractions.     

Innexin and pannexin channels and their signaling

Innexins are bifunctional membrane proteins in invertebrates, forming gap junctions as well as non-junctional membrane channels (innexons). Their vertebrate analogues, the pannexins, have not only lost the ability to form gap junctions but are also prevented from it by glycosylation. Pannexins appear to form only non-junctional membrane channels (pannexons). The membrane channels formed by pannexins and innexins are similar in their biophysical and pharmacological properties. Innexons and pannexons are permeable to ATP, are present in glial cells, and are involved in activation of microglia by calcium waves in glia. Directional movement and accumulation of microglia following nerve injury, which has been studied in the leech which has unusually large glial cells, involves at least 3 signals: ATP is the “go” signal, NO is the “where” signal and arachidonic acid is a “stop” signal.     

Distribution of Connexin50 Channels and Hemichannels in Lens Fibers: A Structural Approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Distribution of Connexin50 Channels and Hemichannels in Lens Fibers: A Structural Approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Distribution of connexin50 channels and hemichannels in lens fibers: a structural approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

A simplified method for the rapid isolation of cardiac intercalated discs

A simple and rapid method for the isolation of intercalated discs from a single rat heart is described. The outstanding features of this method are (i) a large quantity of starting material is not necessary, (ii) ultracentrifugation and separating gradient steps are not required, (iii) the disc fraction is relatively pure as verified by electron microscopy, and (iv) the starting tissue is rapidly homogenized into buffer, reducing possible damage to membrane structures after animal death. The fraction is rich in intercellular junctions, with gap junctions, fasciae adherentes and maculae adherentes all appearing well preserved. It should prove a useful starting base for further purification of these junctions and of sarcolemma from a specific portion of the cell surface membrane, namely the major area of structural interaction between adjacent cardiac cells.     

Hormonal regulation of gap junction differentiation

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.     

Intercellular adhesivity and pupal morphogenesis inDrosophila melanogaster

Summary Leg and wing imaginal discs of mature larvae ofDrosophila melanogaster when treated with 0.1% trypsin for 5–10 min underwent a change in shape that closely resembled normal pupal morphogenesis. Simultaneously, the cells of the disc epithelium changed in shape from tall columnar to cuboidal. Colcemid eliminated microtubules but was without effect on the shape of the imaginal discs or their cells. Tryptic digestion reduced non-junctional intercellular adhesivity but septate desmosomes and gap junctions remained intact.It is proposed that the structure of imaginal discs permits the packaging of the anlagen of the adult integument so that they can change shape and replace the larval structures in a brief period. Apparently most of the definitive form of the pupal leg is built into the disc and becomes visible within a few minutes as intercellular adhesivity is changed.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).     

Gap junctions and rhombic particle arrays in the freeze-fractured mouse oocyte-follicle cell complex

Intact follicles as well as defolliculated oocytes of the mouse were studied by freeze-fracture electron microscopy. In intact follicles the oocyte plasma membrane shows two prominent types of intra-membrane particle array:gap junctions and yet undescribed rhombic particle arrays. The gap junctions vary in size (from 5 to 500 IMPs) and shape. Occasionally they are organized in so-called formation plaques. The rhombic particle arrays consist of 25 IMPs on an average, the IMP diameter is 10.5 nm, the mean IMP distance is 19.8 nm and the acute angle in the array is 81.3 degrees. After defolliculation the gap junctions disassemble and change transiently into linear IMP arrays. The rhombic particle arrays persist indicating that they are of a non-junctional nature. The possible function of the rhombic particle arrays is discussed in relation to similar membrane specializations in excitable cells.     

Junctional complexes, perineurial and glia-axonal relationships and the ensheathing structures of the insect nervous system; A comparative study using conventional and freeze-cleaving techniques

When the ventral nerve cord of the locust or cockroach is examined after freeze-cleaving, the fine structure of the various tissues comprising the ganglia and connectives do not differ radically from conventional glutaraldehyde-fixed, sectioned material. The extracellular spaces, the extent of which is of considerable importance in calculating cation reservoirs, appear very similar in dimension after freezing unfixed tissue and after thin sectioning fixed material. Further comparisons between the structures revealed by freeze-cleaving briefly-fixed and unfixed nerve cords are described. The plane of freeze-cleavage along membrane faces yields new information about the extent of intercellular junctions and both the hemi-desmosomes and gap junctions in these tissues are found to be macular in outline. Evidence from the penetration of extracellular tracers as well as freeze-cleaving shows that the gap junctions are composed of subunits of the larger of the two types known so far to exist. Septate desmosomes are shown by both methods to be relatively disorganized in nature. ‘Tight’ junctions (zonulae or fasciae occludentes), found between perineurial cells and considered to be the morphological basis of the blood-brain barrier in insects, differ in freeze-cleave preparations from their vertebrate equivalent, being rather simpler in structure. Although basically very alike, a few relatively subtle differences are apparent in comparisons of insect tissues prepared by the two methods; these include variations in the cross-sectional appearance of the collagen-like fibrils in the neural lamella and differences in the diameter of microtubules.     

Cell junctions and other membrane specializations in the ocellus of the wasp,Paravespula Germanica L. (Hymenoptera : Vespidae)

Five types of cell contacts and other membrane specializations were found in the ocellus of the adult wasp, Paravespula germanica L. (Hymenoptera : Vespidae), based on freeze-fracture replicas and thin sections.Septate junctions alongside small gap junctions are present between iris cells and between corneagenous cells. Gap junctions are sometimes observed between glial cell processes. Photoreceptor cells and glial cells are frequently connected by scalariform junctions. Tight junction-like structures are found on receptor-cell membranes near rhabdomeric microvilli. Desmosomes are widespread in the ocellus, connecting iris cells, corneagenous cells, receptor cells, and glial cell processes. Desmosomes are found next to septate junctions.Glial membranes connected to receptor cells have a non-junctional type of membrane specializations, consisting of intramembraneous particles arranged in a rhombic pattern. Interestingly, both particle arrays and scalariform junctions are often adjacent to each other. Furthermore, a conspicuous modification of the cell surface in freeze cleaved cells is seen between adjacent glial cells intermediating two receptor cells.     

Immunocytochemical,electrophoresis, and immunoblot analysis of Heliothis virescens gap junctions isolated in the presence and absence of protease inhibitors

Gap junction-enriched fractions were prepared from larvae of the tobacco budworm Heliothis virescens using the NaOH procedure in the presence or absence of protease inhibitors and were analyzed by SDS-PAGE, immunoblotting and EM immunocytochemistry. Protease inhibitor fractions contained a 48-kDa protein in addition to the 10 proteins in fractions with and without inhibitors. Three polyclonal antibodies were used as probes for gap junction plaques and proteins: R16, against an 40-kDa candidate gap junction protein from Drosophila melanogaster; R17, against the 40-kDa candidate gap junction protein from H. virescens; and R18AP, an affinity purified antibody against a consensus sequence of N-terminal amino acids 2–21 of the H. virescens 40-kDa protein. R16, R17, and R18AP stain the 40- and 48-kDa proteins, R16 and R18AP stain a 64-kDa protein, and R16 stains an 30-kDa protein in the absence of inhibitors. Inclusion of protease inhibitors had no effect on gap junction ultrastructure. R16 and R17 label gap junction plaques in crude membrane and NaOH fractions, whereas R18AP exhibits only a low level of reactivity with gap junctions in crude membrane fractions and none with gap junctions in NaOH fractions. The results show that the 30-, 40-, 48- and 64-kDa proteins are immunologically related and are associated with gap junctions in H. virescens, the N-terminus of the 40-kDa protein is relatively inaccessible or easily lost, and the 48-kDa protein is protease-sensitive.     

Immunolocalization of an extracellular domain of connexin43 in rat heart gap junctions.

Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.     

Nature of plasmalemmal functional "hemichannels"

The molecular identity of the protein forming "hemichannels" at non-junctional membranes is disputed. The family of gap junction proteins, innexins, connexins, and pannexins share several common features, including permeability characteristics and sensitivity to blocking agents. Such overlap in properties renders the identification of which of these protein species actually establishes the non-junctional membrane conductance and permeability quite complicated, especially because in vertebrates pannexins and connexins have largely overlapping distributions in tissues. Recently, attempts to establish criteria to identify events that are "hemichannel" mediated and those to allow the distinction between connexin- from pannexin-mediated events have been proposed. Here, I present an update on that topic and discuss the most recent findings related to the nature of functional "hemichannels" focusing on connexin43 and pannexin1. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures

The topological organization of the major rat liver gap junction protein has been examined in intact gap junctions and gap junction-derived single membrane structures. Two methods, low pH and urea at alkaline pH, were used to "transform" or "split" double membrane gap junctions into single membrane structures. Low pH treatment "transforms" rat liver gap junctions into small single membrane vesicles which have an altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after digestion with L-1-to-sylamido-2-phenylethylchloromethyl ketone-trypsin. Alkaline pH treatment in the presence of 8 M urea can split isolated rat liver gap junctions into single membrane sheets which have no detectable structural alteration or altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after proteolytic digestion, suggesting that these single membrane sheets may be useful for topological studies of the gap junction protein. Proteolytic digestion studies have been used to localize the carboxyl terminus of the molecule on the cytoplasmic surface of the intact gap junction. However, the amino terminus does not appear to be accessible to proteases or to interaction with an antibody that is specific for the amino-terminal region of the molecule in intact or split gap junctions. Binding of antibodies, that block junctional channel conductance, can be eliminated by proteolytic digestion of intact gap junctions, suggesting that all antigenic sites for these antibodies are located on the cytoplasmic surface of the intact gap junction. In addition, calmodulin gel overlays indicate that at least two calmodulin binding sites exist on the cytoplasmic surface of the junctional protein. The information generated from these studies has been used to develop a low resolution two-dimensional model for the organization of the major rat liver gap junctional protein in the junctional membrane.     

Effect of cholesterol deficiency on the composition of phospholipids and fatty acids of the housefly, Musca domestica

The housefly larvae were grown in the aseptic diet containing 0.56 μmole cholesterol/g wet weight of diet (control) and 0.05 μmole cholesterol/g wet weight of diet (deficient). The effects of cholesterol deficiency upon the phospholipid composition and fatty acids of the total phospholipid and triglyceride fractions from the lipid extract of the various larval tissues, whole larva, and in both sexes of adults 4 days after eclosion were examined. The total sterol and phospholipid contents (expressed relative to the wet weight of the insect) of the control and deficient insects at the larval and adult stages were analysed and molar ratios compared. The results suggest that cholesterol deficiency reduced the free sterol content of the larvae and adult insects to approximately 25% of the content of the control insects. However, cholesterol deficiency did not effect the phospholipid content during larval and adult stages when compared to that of control insects. Though the larvae reared on the cholesterol deficient diets did not show a profound alteration in the phospholipid composition, a marked increase in the ratio of phosphatidylcholine to phosphatidylethanolamine of the larval fat body and composite gut fraction were noticed. The cholesterol deficiency induced significant changes in the fatty acid composition of the phospholipid fraction of the insect. The ratio of unsaturated fatty acids to saturated fatty acids of the phospholipid fractions decreased significantly due to cholesterol deficiency in the whole larvae and in both sexes of adult flies. The data indicates that cholesterol deficient insects compensated for the lack of cholesterol by increasing saturated fatty acids preferentially in the phospholipid fraction of the lipids for the maintenance of proper membrane fluidity.