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D A Goodenough

Publications and source records attributed to D A Goodenough.

At least 55 records · Page 3Linked to original sources
PubMed1994

Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein.

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160-kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase-like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs-large-1 (dlg) gene of Drosophila, and a synapse-associated protein from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double-label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction.

Amino Acid Sequence↗
PubMed1994

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins.

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.

Amino Acid Sequence↗
PubMed1994

Molecular cloning and functional characterization of chick lens fiber connexin 45.6.

The avian lens is an ideal system to study gap junctional intercellular communication in development and homeostasis. The lens is experimentally more accessible in the developing chick embryo than in other organisms, and chick lens cells differentiate well in primary cultures. However, only two members of the connexin gene family have been identified in the avian lens, whereas three are known in the mammalian system. We report here the molecular cloning and characterization of the third lens connexin, chick connexin45.6 (ChCx45.6), a protein with a predicted molecular mass of 45.6 kDa. ChCx45.6 was encoded by a single copy gene and was expressed specifically in the lens. There were two mRNA species of 6.4 kilobase (kb) and 9.4 kb in length. ChCx45.6 was a functional connexin protein, because expression in Xenopus oocyte pairs resulted in the development of high levels of conductance with a characteristic voltage sensitivity. Antisera were raised against ChCx45.6 and chick connexin56 (ChCx56), another avian lens-specific connexin, permitting the examination of the distribution of both proteins. Immunofluorescence localization showed that both ChCx45.6 and ChCx56 were abundant in lens fibers. Treatment of lens membranes with alkaline phosphatase resulted in electrophoretic mobility shifts, demonstrating that both ChCx45.6 and ChCx56 were phosphoproteins in vivo.

Amino Acid Sequence↗
PubMed1993

Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER.

Connexin43 (Cx43) is an integral plasma membrane protein that forms gap junctions between vertebrate cells. We have used sucrose gradient fractionation and chemical cross-linking to study the first step in gap junction assembly, oligomerization of Cx43 monomers into connexon channels. In contrast with other plasma membrane proteins, multisubunit assembly of Cx43 was specifically and completely blocked when endoplasmic reticulum (ER)-to-Golgi transport was inhibited by 15 degrees C incubation, carbonyl cyanide m-chloro-phenylhydrazone, or brefeldin A or in CHO cell mutants with temperature-sensitive defects in secretion. Additional experiments indicated that connexon assembly occurred intracellularly, most likely in the trans-Golgi network. These results describe a post-ER assembly pathway for integral membrane proteins and have implications for the relationship between membrane protein oligomerization and intracellular transport.

Animals↗
PubMed1993

Gap junctions and tissue business: problems and strategies for developing specific functional reagents.

The complex and overlapping tissue distribution of different members of the gap junctional connexin protein family is reviewed. Intermixing of different connexins in the building of intercellular channels and translational and posttranslational regulation of gap junctional channels add additional challenges to the interpretation of the possible functions played by gap junction-mediated intercellular communication in tissue business.

Animals↗
PubMed1993

Posttranslational phosphorylation of lens fiber connexin46: a slow occurrence.

PURPOSE: To study in both in situ and primary cultures the posttranslational phosphorylation of connexin46 (Cx46), one of two members of the connexin family of gap junction proteins expressed by lens fibers. METHODS: Phosphatase digestion, gel electrophoresis, cell culture, organ culture, immunoprecipitation, metabolic labeling, and phosphoamino acid analysis were the methods used in this study. RESULTS: Cx46 immunoprecipitated from either rat or bovine lenses resulted in a shift to a more rapidly migrating species. During rat embryonic development, the more rapidly migrating, nonphosphorylated form of Cx46 was prevalent at 15 days gestation; as development progressed, there was a loss of the nonphosphorylated form with a concomitant increase in the phosphorylated form, such that by 28 days after birth only the phosphorylated form was detectable. The rate of posttranslational phosphorylation was very slow compared to previously measured rates for connexin43. Primary cultures of rat embryonic lens epithelial cells, which contained differentiating lentoids, were labeled with 35S-methionine and chased for 8 days. Very low levels of Cx46 were detectable, and none of this labeled material shifted to the slower mobility during the 8-day chase period. Similarly, in organ culture of bovine lenses, Cx46 could be labeled with 35S-methionine, but the immunoprecipitated material remained in the rapidly migrating form for 1 week, the longest time measured. This immunoprecipitated material was shown to be serine-phosphorylated, which was insufficient to cause the electrophoretic mobility shift. CONCLUSIONS: There are low levels of Cx46 synthesis and phosphorylation in rat embryo lens primary cultures and a slow rate of phosphorylation of Cx46 in bovine organ cultures.

Animals↗
PubMed1992

Ion channels in single bilayers induced by rat connexin32.

The gap junction channel mediates an important form of intercellular communication, but its detailed study is hindered by inaccessibility in situ. We show here that connexin32, the major protein composing junctional channels in rat liver, forms ion channels in single bilayer membranes. The properties of these reconstituted connexin32 channels are characterized and compared with those of gap junction channels. The demonstration that connexin32 forms channels in single membranes has implications for assembly and regulation of junctional channels, and permits detailed study of the gating, permeability and modulation of this channel-forming protein.

Animals↗
PubMed1992

The crystalline lens. A system networked by gap junctional intercellular communication.

The vertebrate eye lens is a solid cyst of cells which grows throughout life by addition of new cells at the surface. The older cells, buried by the newer generations, differentiate into long, prismatic fibers, losing their cellular organelles and filling their cytoplasms with high concentrations of soluble proteins, the crystallins. The long-lived lens fibers are interconnected by gap junctions, both with themselves and with an anterior layer of simple cuboidal epithelial cells at the lens surface. This network of gap junctions joins the lens cells into a syncytium with respect to small molecules, permitting metabolic co-operation: intercellular diffusion of ions, metabolites, and water. In contact with nutrients at the lens surface, the epithelial cells retain their cellular organelles, and are able to provide the metabolic energy to maintain correct ion and metabolite concentrations within the lens fiber cytoplasms, such that the crystallins remain in solution and do not aggregate (cataract). Gap junctions are formed by a family of integral membrane channel-forming proteins called connexins. Gap junctions between lens epithelial cells are composed of a connexin which is common between many different cell types, notably myocardial cells and connective tissue fibroblasts. The gap junctions between epithelial cells and lens fibers have not yet been biochemically characterized. The gap junctions formed between lens fibers are composed of at least two different connexins, one of which has not been detected between other cell types. The unusual physiology and longevity of the lens fibers may require the special set of connexins which are found joining these cells.

Animals↗
PubMed1992

Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70.

The crystalline lens is an attractive system to study the biology of intercellular communication; however, the identity of the structural components of gap junctions in the lens has been controversial. We have cloned a novel member of the connexin family of gap junction proteins, Cx50, and have shown that it is likely to correspond to the previously described lens fiber protein MP70. The N-terminal amino acid sequence of MP70 closely matches the sequence predicted by the clone. Cx50 mRNA is detected only in the lens, among the 12 organs tested, and this distribution is indistinguishable from that of MP70 protein. A monoclonal antibody directed against MP70 and an anti-Cx50 antibody produced against a synthetic peptide identify the same proteins on western blots and produce identical patterns of immunofluorescence on frozen sections of rodent lens. We also show that expression of Cx50 in paired Xenopus oocytes induces high levels of voltage-dependent conductance. This indicates that Cx50 is a functional member of the connexin family with unique physiological properties. With the cloning of Cx50, all known participants in gap junction formation between various cell types in the lens are available for study and reconstitution in experimental systems.

Animals↗
PubMed1991

Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes.

Gap junctions are composed of a family of structural proteins called connexins, which oligomerize into intercellular channels and function to exchange low molecular weight metabolites and ions between adjacent cells. We have cloned a new member of the connexin family from lens cDNA, with a predicted molecular mass of 46 kD, called rat connexin46 (Cx46). Since a full-length cDNA corresponding to the 2.8-kb mRNA was not obtained, the stop codon and surrounding sequences were confirmed from rat genomic DNA. The RNA coding for this protein is abundant in lens fibers and detectable in both myocardium and kidney. Western analysis of both rat and bovine lens membrane proteins, using the anti-MP70 monoclonal antibody 6-4-B2-C6 and three anti-peptide antibodies against Cx46 demonstrates that Cx46 and MP70 are different proteins. Immunocytochemistry demonstrates that both proteins are localized in the same lens fiber junctional maculae. Synthesis of Cx46 in either reticulocyte lysate or Xenopus oocytes yields a 46-kD polypeptide; all anti-Cx46 antisera recognize a protein in rat lens membranes 5-10 kD larger, suggesting substantive lenticular posttranslational processing of the native translation product. Oocytes that have synthesized Cx46 depolarize and lyse within 24 h, a phenomenon never observed after expression of rat connexins 32 or 43 (Cx32 and Cx43). Lysis is prevented by osmotically buffering the oocytes with 5% Ficoll. Ficoll-buffered oocytes expressing Cx46 are permeable to Lucifer Yellow but not FITC-labeled BSA, indicating the presence of selective membrane permeabilities. Cx43-expressing oocytes are impermeable to Lucifer Yellow. Voltage-gated whole cell currents are measured in oocytes injected with dilute concentrations of Cx46 but not Cx43 mRNA. These currents are activated at potentials positive to -10 mV. Unlike other connexins expressed in Xenopus oocytes, these results suggest that unprocessed Cx46 induces nonselective channels in the oolemma that are voltage dependent and opened by large depolarizations.

Amino Acid Sequence↗
PubMed1991

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

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.

Biological Transport↗
PubMed1990

Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation.

Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with 32P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.

Amino Acid Sequence↗
PubMed1990

Correlation analysis of gap junction lattice images.

Fourier averages of connexon images computed from low-irradiation electron micrographs of isolated negatively stained gap junction domains exhibited differences in stain distribution and connexon orientation. To analyze these polymorphic structures, correlation averaging methods were applied to images from negatively stained and frozen-hydrated specimens. For the negatively stained specimens, separate averages over two subsets of connexons with differing degrees of stain accumulation in the axial channel were obtained. Two populations of connexons with opposite skew orientations were distinguishable within a single junctional domain of a frozen-hydrated specimen. Correlation maps calculated using the left- and right-skewed references showed that the selected connexons tend to locally cluster. Using correlation methods to analyze packing disorder in a typical connexon lattice, we estimated the root-mean-square variation in the nearest neighbor pair separation to be approximately 11% of the lattice constant. Displacements of the connexons relative to each other increased with increasing pair separation in the lattice, rather like a liquid, although long-range orientation order was conserved as in a crystal. These results support the hypothesis that the hexagonal ordering of the connexons results from short-range repulsive forces.

Biophysical Phenomena↗
PubMed1990

Diffraction diagnosis of protein folding in gap junction connexons.

To diagnose the regular polypeptide conformation in gap junction membranes, the x-ray intensities diffracted from oriented specimens have been separated into a modulated component due to the coherently ordered portion of the channel-forming pairs of connexon hexamers and a diffuse component due to the disordered parts. The spherically averaged ordered protein diffraction, in the resolution range 15-4 A, was compared with intensity curves calculated from the Fourier transforms of proteins representative of the major tertiary structural classes. From this comparison the alpha-helical content of the ordered portion of the connexon was estimated to be approximately 60%. Calculation of cylindrically averaged patterns for oriented distributions of alpha-helical and beta-sheet proteins demonstrated that the ratio of the modulated diffracted intensity near 5 A spacing on the meridian and 10 A spacing on the equator observed from the gap junctions can be accounted for by alpha-helical segments inclined relative to the connexon axis. Model dimers of connexonlike hexamers were constructed from alpha-helix bundle proteins to correlate features in the calculated diffraction patterns with the model parameters. On the basis of these correlations, the ordered gap junction diffraction data indicate that alpha-helical segments centered at 38 A from the midplane of the gap have a mean radial location approximately 24 A from the hexamer axis, and an axial projected length of approximately 35 A. Thus, these alpha-helical segments traverse the hydrocarbon core of the lipid bilayer, as expected for the four hydrophobic sequences of the connexin molecule.

Animals↗
PubMed1990

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

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.

Animals↗
PubMed1989

Cloning and expression of a Xenopus embryonic gap junction protein.

Gap junctions in the early amphibian embryo may play a fundamental role in the regulation of differentiation by mediating the cell-to-cell transfer of chemical signals. A complementary DNA encoding a gap junction present in Xenopus oocytes and early embryos has now been cloned and sequenced. This protein sequence is homologous to the well-characterized gap junction structural proteins rat connexin32 and connexin43. RNA blot analysis of total Xenopus oocyte RNA showed hybridization to a single 1.6-kilobase band. This messenger RNA is abundant in oocytes, decreases to levels below the sensitivity of our assay by stage 15 (18 hours), and is not detectable in RNA from a number of adult organs. To confirm that the oocyte cDNA encodes a gap junction channel, the protein was over expressed in Xenopus oocytes by injection of RNA synthesized in vitro. Pairs of RNA-injected oocytes formed many more time- and voltage-sensitive cell-cell channels than water-injected pairs.

Amino Acid Sequence↗