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D A Goodenough

Publications and source records attributed to D A Goodenough.

101 records · Page 6Linked to original sources
PubMed1976

Tight and gap junctions in a vertebrate inner ear.

The auditory organ of the alligator lizard has been investigated with the transmission electron microscope using methods which distinguish between tight and gap junctions. There is a continuous zone of tight junctions located near the endolymphatic surface of the organ forming a boundary between the endolymph in scala media and the interstitial spaces between the cells. No such tight junctions were observed between the perilymph of scala tympani and the interstitial fluid within the organ. Small gap junctions occur between hair cells and supporting cells and large gap junctions occur between adjacent supporting cells. The locations of the tight junctions suggest that the composition of the intercellular fluid in the receptor organ is probably more like perilymph than like endolymph. The presence of gap junctions between hair cells and supporting cells provides a possible morphological basis for the occurrence of intracellular responses to sound in supporting cells, and for elctric coupling of receptor cells.

Animals↗
PubMed1976

In vitro formation of gap junction vesicles.

A method is described that uses trypsin digestion combined with collagenase-hyaluronidase which produces a population of gap junction vesicles. The hexagonal lattice of subunits ("connexons") comprising the gapjunctions appears unaltered by various structural criteria and by buoyant density measurements. The gap junction vesciles are closed by either a single or a double profile of nonjunctional "membrane," which presents a smooth, particle-free fracture face. Horseradish peroxidase and cytochrome c studies have revealed that about 20% of the gap junction vesicles are impermeable to proteins 12,000 daltons or larger. The increased purity of the trypsinized junction preparation suggests that one of the disulfide reduction products of the gap-junction principal protein may be a nonjunctional contaminating peptide. The gap junction appears to be composed of a single 18,000-dalton protein, connexin, which may be reduced to a single 9,000-dalton peak. The number of peptides in this reduced peak are still unknown.

Animals↗
PubMed1976

The structure and permeability of isolated hepatocyte gap junctions.

The ultrastructure of the gap junction may be visualized in both in situ and isolated preparations by using a variety of electron microscope techniques. The junction is composed of a lattice of subunits, called connexons, which show variable degrees of packing into a hexagonal lattice depending on a variety of poorly understood conditions. In general, it appears that more uncoupled and "dead" the junction, the more regular and condensed the hexagonal lattice becomes. It is not yet known whether these are "postmortem" changes or physiologically active and reversible changes involved in regulation of junctional permeability. Using a variety of techniques, it can be seen that the connexon extends completely across the junctional membranes, from the cytoplasmic surface of one cell to the cytoplasmic surface of the other, spanning the 2-nm "gap" between the apposed junctional membranes. Thus it is possible to implicate the connexon as a permeability channel from cytoplasm to cytoplasm, although the hydrophilic pore through the center of the connexon has not yet been demonstrated to span the full junction thickness. X-ray diffraction experiments support these conclusions, and the excellent correlation between the electron microscope and X-ray diffraction data lends great confidence to the interpretations of gap junction structure presented thus far. These data are summarized in the drawing in Figure 17. This is a scale drawing of two connexons, each of which is imagined to be composed of a dimer of hexamers. It must be emphasized that as yet there is no direct evidence for a sixfold symmetry within the connexon. Of special interest now are the types of protein-protein interactions that hold the two halves of the connexon together across the 2-nm gap and the lateral interactions between the connexons at the level of the lipid bilayers.

Animals↗
PubMed1975

Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction.

A structural and biochemical study is presented concerning the agglutination of gametic flagella, the initial step in the mating reaction of Chlamydomonas reinhardtii. An alteration in the distribution of the intramembranous particles revealed by freeze-fracturing of flagella membranes is shown to accompany gametic differentiation in both mating types. The isolation and electrophoretic analysis of flagellar membranes and mastigonemes are reported; no electrophoretic differences can be detected when the membrane or mastigoneme glycoproteins from vegative and gametic cells are compared, nor when glycoproteins from the two mating types are compared, and no novel polypeptides are present in gametic preparations. The membrane vesicles, after they are freed of mastigonemes by sedimentation through a discontinuous sucrose gradient, are extremely active as an isoagglutinin, indicating a direct involvement of the membrane in the mating reaction.

Agglutination↗
PubMed1975

The structure of cell membranes involved in intercellular communication.

The molecular structure of mouse hepatocyte gap junctions is investigated with corrlated electron microscopy, biochemistry, and x-ray diffraction technics. These studies reveal that the gap junction is composed of a hexagonal lattice of protein subunits, connexons, which pierce the hydrophobic membrane and establish a structural basis for intercellular hydrophilic channels or pores. By digesting liver-cell membranes with trypsin, a preparation of open- and closed-gap junction vesicles can be generated; this preparation will permit direct permeability measurements across the gap junction membranes in an in-vitro system.

Animals↗
PubMed1974

The splitting of hepatocyte gap junctions and zonulae occludentes with hypertonic disaccharides.

Mouse livers were perfused in situ through the portal vein with the disaccharides sucrose, lactose, maltose, and cellobiose in hypertonic concentrations (0.5 M). This treatment resulted in plasmolysis of the hepatocytes and splitting of the gap junctions and zonulae occludentes. The junctions split symmetrically, leaving a half-junction on each of the two separated cells. The process of junction splitting is followed using the freeze-fracture technique, since the junctional membranes are indistinguishable from the nonjunctional membranes in thin sections once the splitting occurs. The split junctions are also studied using the freeze-etch technique, allowing a view of the gap junction extracellular surface normally sequestered within the 2-nm "gap." The monosaccharides sorbitol and mannitol did not split the junctions during the times studied (2 min), but substitution of the chloride ion with propionate in the perfusion mixture did result in junction splitting. An envelope of morphologically distinct particles surrounding freeze-fractured gap junctions is also described.

Animals↗
PubMed1973

Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia.

Epithelia vary with respect to transepithelial permeability. In those that are considered "leaky", a large fraction of the passive transepithelial flux appears to follow the paracellular route, passing across the zonulae occludentes and moving down the intercellular clefts. In "tight" epithelia, the resistance of the paracellular pathway to passive flux is greatly increased. To see whether differences in the morphology of the zonula occludens could contribute to this variability in leakiness among epithelia, replicas of zonulae occludentes in freeze-fractured material from a variety of tight and leaky epithelia were examined. The junctions appear as a branching and anastomosing network of strands or grooves on the A and B membrane fracture faces, respectively. It was found that the zonula occludens from a "very leaky" epithelium, the proximal convoluted tubule of the mouse kidney, is extremely shallow in the apical-basal direction, consisting in most places of only one junctional strand. In contrast, the "very tight" frog urinary bladder exhibits a zonula occludens that is relatively deep (>0.5 microm) in the apical-basal direction, and consists of five or more interconnected junctional strands interposed between luminal and lateral membrane surfaces. Epithelia of intermediate permeabilities exhibited junctions with intermediate or variable morphology. Toad urinary bladder, mouse stomach, jejunum, and distal tubule, rabbit gallbladder, and Necturus kidney and gallbladder were also examined, and the morphological data from these epithelia were compared to physiological data from the literature.

Animals↗
PubMed1972

The isolation of mouse hepatocyte gap junctions. Preliminary chemical characterization and x-ray diffraction.

A method is reported for isolating a preparation of hepatic gap junctions from the mouse. The method involves a collagenase digestion, treatment with the detergent Sarkosyl NL-97, and ultrasonication, followed by sucrose gradient ultracentrifugation. A run with 36 animals yields 0.1-0.5 mg protein. Electron microscopy with thin-sectioning and negative staining techniques reveals that the final pellet is a very pure preparation of gap junctions, accompanied by a small amount of amorphous contamination. Polyacrylamide-gel electrophoresis of sodium dodecyl sulfate (SDS)-solubilized material shows one major protein in the junction, with an apparent mol wt of 20,000, and two minor components. Thin-layer chromatography demonstrates one major and one minor phospholipid, and some neutral lipid. Low-angle X-ray diffraction of wet and dried specimens show reflections which index on an 86 A center-to-center hexagonal lattice, corresponding closely to electron microscope data. Dried specimens also show a lamellar diffraction, corresponding to the total profile thickness of the junction (150 A).

Animals↗
PubMed1971

The permeability of isolated and in situ mouse hepatic gap junctions studied with enzymatic tracers.

We have studied the effects of phospholipase C from Clostridium welchii on gap junctions in the intact mouse liver and in a junction-rich fraction prepared from mouse liver. Treatment of the isolated junctions results in the disappearance of both the 20 A gap and of the polygonal lattice visible with lanthanum. The junctions are morphologically unaltered, however, when whole livers are perfused with phospholipase via the portal vein. These results suggest that extracellular phospholipase cannot diffuse into the junctional area, but that the enzyme may affect structures within the gap from its cytoplasmic surfaces which become exposed in the isolated preparations. Horseradish peroxidase, which has physical dimensions similar to those of Clostridium phospholipase is also denied access to the 20 A gap in whole liver, while peroxidase reaction product can be seen in the gap in isolated preparations. Beef liver catalase, however, a tracer molecule much larger than peroxidase, cannot penetrate even in isolated fractions. If the cytoplasmic approaches to the gap junction used by peroxidase and phospholipase are available in vivo, and have not been created during the process of mechanical isolation, they may play a role in cell-to-cell passage of molecules larger than ions.

Animals↗
PubMed1970

A fine structural analysis of intercellular junctions in the mouse liver.

Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90-100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.

Acetates↗