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D A Goodenough

Publications and source records attributed to D A Goodenough.

18 recordsLinked to original sources
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↗
PubMed1979

Structural states of myelin observed by x-ray diffraction and freeze-fracture electron microscopy.

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.

Animals↗
PubMed1979

Lens gap junctions: a structural hypothesis for nonregulated low-resistance intercellular pathways.

Structural evidence is presented which suggests that gap junctions between lens fibers are adapted to remain in a low-resistance physiological state, under conditions which switch gap junctions in other tissues to a high-resistance state. The lens gap junction subunits (connexons) do not crystallize in the membrane plane in response to fixation, anoxia, lens damage, or homogenization and isolation. Rapid freezing experiments (Raviola et al.) suggest connexon resistance. Freshly homogenized liver cytoplasm does not contain an assayable factor which can crystallize connexons in lens gap junctions. Polyacrylamide gel electrophoresis of enrighed preparations of isolated lens junctions reveals a 27 +/- 2 kilodalton principal polypeptide which is similar in electrophoretic mobility to one of the principal polypeptides resolved in gels of isolated hepatocyte gap junctions. These results indicate that the whole lens may be extremely vulnerable to surface injury, perhaps even to injury of a single lens fiber.

Animals↗
PubMed1978

Flagellar membrane agglutination and sexual signaling in the conditional GAM-1 mutant of Chlamydomonas.

The temperature-sensitive gametogenesis-defective mutant, gam-1 is sex-limited, expressed only in mating type minus (mt-), and can sexually agglutinate but not fuse at the restrictive temperature (35 degrees C) with gametes of wild type (wt) mt+. Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gam-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35 degrees C, each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gam-1 flagella. The two mutant phenotypes displayed by gam-1 have been denoted gam-1-I and gam-1-II. An agglutination reaction involving gam-1-I cells does not result in activation of the wt mt+ mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-1-II cells, but because the gam-1 mt- mating sturcture still fails to activate, cell fusion is precluded. We conclude that the gam-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.

Agglutination↗
PubMed1977

Membrane particle arrays associated with the basal body and with contractile vacuole secretion in Chlamydomonas.

Freeze-fracture replicas reveal that five distinct types of intramembranous particle arrays coexist within a small sector of the C. reinhardtii cell flagellar membrane. Of these, three are newly described in this report. (a) Flagellar bracelets, which encircle the flagellar bases, appear to be intrinsically ordered strands of particles of unknown function. (b) Strut arrays, representing nine sites where the basal body attaches to the membrane, appear to serve a mechanical function. (c) Contractile vacuole arrays, which develop into circular plaques of particles, appear to serve as "membrane gates" through which water is discharged from the cell.

Cell Membrane↗
PubMed1977

Membrane differentiations at sites specialized for cell fusion.

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.

Cell Fusion↗
PubMed1977

Gap junction structures. I. Correlated electron microscopy and x-ray diffraction.

X-ray crystallographic methods and electron microscope image analysis have been used to correlate the structure and the chemical composition of gap junction plaques isolated intact from mouse liver. The requirement that the interpretations of X-ray, electron microscope, and chemical measurements be consistent reduces the uncertainties inherent in the separate observations and leads to a unified picture of the gap junction structures. Gap junctions are built up of units called connexons that are hexagonally arrayed in the pair of connected cell membranes. X-ray diffraction and electron microscope measurements show that the lattice constant of this array varies from about 80 to 90 A. Analysis of electron micrographs of negatively stained gap junctions shows that there is significant short range disorder in the junction lattice. even though the long range order of the array is remarkably regular. Analysis of the disorder provides information about the nature of the intermolecular forces that hold the array together.

Animals↗
PubMed1977

Gap junction structures. II. Analysis of the x-ray diffraction data.

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.

Animals↗
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↗