Biomedical subjects

D A Goodenough

Publications and source records attributed to D A Goodenough.

At least 73 records · Page 4Linked to original sources

PubMed1989

Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues.

Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.

Animals↗

PubMed1988

Gap junction structures. VIII. Membrane cross-sections.

Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.

Animals↗

PubMed1988

Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

Animals↗

PubMed1988

Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions.

The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.

Amino Acid Sequence↗

PubMed1988

Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells.

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.

Antibodies, Monoclonal↗

PubMed1988

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219-232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).

Animals↗

PubMed1987

Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions.

Thin section electron microscopy reveals two different types of membrane interactions between the fiber cells of bovine lens. Monoclonal antibodies against lens membrane protein MP70 (Kistler et al., 1985, J. Cell Biol., 101:28-35) bound exclusively to the 16-17-nm intercellular junctions. MP70 localization was most dramatic in the lens outer cortex and strongly reduced deeper in the lens. In contrast, the 12-nm double membrane structures and single membranes were consistently unlabeled. In freeze-fracture replicas with adherent cortical fiber membranes, MP70 was immunolocalized in the junctional plaques which closely resemble the gap junctions in other tissues. MP70 is thus likely to be associated with intercellular communication in the lens.

Animals↗

PubMed1987

Connexin43: a protein from rat heart homologous to a gap junction protein from liver.

Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.

Amino Acid Sequence↗

PubMed1986

Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell.

Lens epithelial cells communicate with two different cell types. They communicate with other epithelial cells via gap junctions on their lateral membranes, and with fiber cells via junctions on their apices. We tested independently these two routes of cell-cell communication to determine if treatment with a 90% CO2-equilibrated medium caused a decrease in junctional permeability; the transfer of fluorescent dye was used as the assay. We found that the high-CO2 treatment blocked intraepithelial dye transfer but not fiber-to-epithelium dye transfer. The lens epithelial cell thus forms at least two physiologically distinct classes of gap junctions.

Animals↗

PubMed1986

Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."

Animals↗

PubMed1986

Rat lens cultures: MIP expression and domains of intercellular coupling.

A simple means of establishing a differentiating rat lens culture system is presented which exhibits a high degree of both morphologic and biochemical differentiation along lens-specific lines. Morphological differentiation includes cell enlargement, displacement from the cell substratum, and the loss of intracellular organelles. Biochemical differentiation includes the de novo synthesis of the Main Intrinsic Polypeptide (MIP), a putative fiber cell junctional polypeptide, as well as the synthesis of the beta and gamma crystallins. Mapping of domains of intercellular communication as defined by the spread of intracellularly injected Lucifer Yellow CH and electrical coupling, are compared to the distribution of biochemically and morphologically differentiated cells.

Animals↗

PubMed1985

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains.

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.

Animals↗

PubMed1985

Gap junction structures after experimental alteration of junctional channel conductance.

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

Animals↗

PubMed1984

Gap junction structures. V. Structural chemistry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility.

X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 A. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 A into the membrane along the channel. Apparently the aqueous channel, 8 to 10 A in radius for most of its length, is narrowed or blocked by a small feature about 50 A from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 A from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 Mr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 A from the 6-fold axis.

Animals↗

PubMed1984

Gap junction structures. VI. Variation and conservation in connexon conformation and packing.

Correlation of structural changes in isolated gap junctions with the mechanism of channel gating is complicated by the effects of isolation procedures and the lack of a direct functional assay. The effect of variations in the isolation procedure are examined by comparison of the structures of gap junctions isolated by different protocols. X-ray diffraction data from over two hundren specimens are compared to provide a basis for identification of invariant aspects of the connexon structure and variable properties related either to functional switching or experimental modifications. We discuss the relationship between subunit tilt, lattice symmetry and packing, and membrane curvature and demonstrate that membrane curvature may be a natural consequence of the structure of the connexons and the patterns of interactions between them.

Animals↗

PubMed1984

Zonulae occludentes in junctional complex-enriched fractions from mouse liver: preliminary morphological and biochemical characterization.

A bile canaliculus-derived preparation containing junctional complexes has been obtained from mouse livers using subcellular fractionation techniques. The junctional complexes include structurally intact zonulae occludentes (ZOs). Extraction of this preparation with the anionic detergent sodium deoxycholate (DOC) left junctional ribbons, the detergent-insoluble zonular remnants of the junctional complexes. When visualized in negative stain electron microscopy, each of these ribbons contained a branching and anastomosing network of fibrils which appears similar to that of ZOs in freeze-fractured whole liver. Comparative measurements of freeze-fracture and negative stain fibril diameters and network densities support this relationship. SDS polyacrylamide gel analysis shows the DOC-insoluble junctional ribbons to be characterized by major polypeptides at 37,000 and at 48,000, with minor bands at 34,000, 41,000, 71,000, 86,000, 92,000, and 102,000. The ZO-containing membrane fractions have been isolated in the presence of EGTA in concentrations and under conditions shown by others to disrupt normal ZO morphology and physiology in whole living epithelia. The network of fibrils visualized in these fractions by negative staining is structurally resistant to treatment with DOC, but is either solubilized or disrupted by N-lauroylsarcosine.

Animals↗

PubMed1983

Gap junction structures. IV. Asymmetric features revealed by low-irradiation microscopy.

Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourier-averaged micrographs of the hexagonal junction lattice show skewed, hexameric connexons with less stain at the threefold axis than at the six indentations between the lobes of the connexon image. These substructural features, not clearly observed previously, are acutely sensitive to irradiation. After an electron dose less than that normally used in microscopy, the image is converted to the familiar doughnut shape, with a darkly stained center and a smooth hexagonal outline, oriented with mirror symmetry in the lattice. Differences in appearance among 25 reconstructed images from our low-irradiation micrographs illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites approximately 34 A from the connexon center, 8 degrees to the right or left of the (1, 1) lattice vector may reveal an intrinsic asymmetric feature of the junction structure. The unexpected skewing of the six-lobed connexon image suggests that the pair of hexagonal membrane arrays that form the junction may not be structurally identical. Because the projected image of the connexon pair itself appears mirror symmetric, each pair may consist of two identical connexon hexamers related by local (noncrystallographic) twofold axes in the junctional plane at the middle of the gap. All connexons may be chemically identical, but their packing in the hexagonal arrays on the two sides of the junction appears to be nonequivalent.

Animals↗

PubMed1983

Preparation, characterization, and localization of antisera against bovine MP26, an integral protein from lens fiber plasma membrane.

Polyclonal antisera were prepared in rabbits using both native and chymotrypsin-digested bovine lens fiber plasma membranes. MP26, the principal protein of lens fiber plasma membranes, and CT20, a chymotryptic fragment of MP26, were isolated electrophoretically and used to purify anti-MP26 and anti-CT20 activity from the respective antisera by affinity chromatography. These affinity-purified antisera were characterized by immunoreplica. Immunofluorescence microscopy localized MP26 on sections of methacrylate-embedded lenses in the lens fiber plasma membranes, but not the lens epithelium. Immunocytochemistry of isolated native or chymotrypsin-digested lens fiber plasma membranes localized both the MP26 and the CT20 only in the nonjunctional plasma membranes, with no detectable activity in the lens fiber junctions themselves. Electron microscopy revealed a second set of pentalaminar profiles, thinner by 4 nm than the lens fiber junctions, which contained demonstrable anti-MP26 and anti-CT20 activity following immunocytochemistry. These results indicate either that MP26 is not a component of the lens fiber junctions, or that significant conformational changes accompany assembly of MP26 into lens fiber junctions, resulting in the masking of MP26 antigenic determinants.

Animals↗