scholarly journals THE SPLITTING OF HEPATOCYTE GAP JUNCTIONS AND ZONULAE OCCLUDENTES WITH HYPERTONIC DISACCHARIDES

1974 ◽  
Vol 61 (3) ◽  
pp. 575-590 ◽  
Author(s):  
Daniel A. Goodenough ◽  
Norton B. Gilula
Keyword(s):  
Portal Vein ◽  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Chloride Ion ◽  
Thin Sections ◽  
Zonulae Occludentes ◽  
The Times

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.

The syneretic lens junction

1984 ◽  
Vol 42 ◽  
pp. 16-19
Author(s):  
J. David Robertson ◽  
M.J. Costello ◽  
T.J. McIntosh
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Membranes ◽  
Freeze Fracture ◽  
Enzymatic Digestion ◽  
Fiber Cell ◽  
Intermembrane Space ◽  
Minor Components ◽  
Thin Sections ◽  
Fiber Cells

The lens of the eye consists of closely adherent greatly elongated flattened narrow fiber cells that are electrically coupled by gap junctions. In thin sections the 100-150 Å intermembrane space usually seen in tissues between adjacent cells is greatly reduced between adjacent fiber cells. Freeze-fracture-etch (FFE) studies have demonstrated gap junctions between fiber cells. Several workers have observed expanses of square crystallinity in fiber cell membranes with a lattice constant of 6-7 nm. This has usually been attributed variously to artifact induced by calcium, pH or proteolytic enzymatic digestion. Square arrays have been seen in isolated fractions of fiber cell membranes prepared with detergents as minor components and dismissed as relatively insignificant and either related or unrelated to gap junctions. Some have regarded them as a form of gap junction.

scholarly journals Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation.

1976 ◽  
Vol 68 (2) ◽  
pp. 173-188 ◽  
Author(s):  
P M Elias ◽  
D S Friend
Keyword(s):  
Vitamin A ◽  
Gap Junctions ◽  
Gap Junction ◽  
De Novo ◽  
Freeze Fracture ◽  
Controlled Study ◽  
Thin Sections ◽  
Tight Junction Formation ◽  
Mucous Metaplasia

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.

scholarly journals Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions.

1988 ◽  
Vol 106 (5) ◽  
pp. 1667-1678 ◽  
Author(s):  
G Zampighi ◽  
M Kreman ◽  
F Ramón ◽  
A L Moreno ◽  
S A Simon
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Structural Changes ◽  
Structural Characteristics ◽  
Freeze Fracture ◽  
Single Particles ◽  
Thin Sections ◽  
Electrophysiological Measurements ◽  
20 Nm ◽  
Junctional Resistance

Gap junctions between crayfish lateral axons were studied by combining anatomical and electrophysiological measurements to determine structural changes associated during uncoupling by axoplasmic acidification. In basal conditions, the junctional resistance, Rj, was approximately 60-80 k omega and the synapses appeared as two adhering membranes; 18-20-nm overall thickness, containing transverse densities (channels) spanning both membranes and the narrow extracellular gap (4-6 nm). In freeze-fracture replicas, the synapses contained greater than 3 X 10(3) gap junction plaques having a total of approximately 3.5 X 10(5) intramembrane particles. "Single" gap junction particles represented approximately 10% of the total number of gap junction particles present in the synapse. Therefore, in basal conditions, most of the gap junction particles were organized in plaques. Moreover, correlations of the total number of gap junction particles with Rj suggested that most of the junctional particles in plaques corresponded to conducting channels. Upon acidification of the axoplasm to pH 6.7-6.8, the junctional resistance increased to approximately 300 k omega and action potentials failed to propagate across the septum. Morphological measurements showed that the total number of gap junction particles in plaques decreased approximately 11-fold to 3.1 X 10(4) whereas the number of single particles dispersed in the axolemmae increased significantly. Thin sections of these synapses showed that the width of the extracellular gap increased from 4-6 nm in basal conditions to 10-20 nm under conditions where axoplasmic pH was 6.7-6.8. These observations suggest that single gap junction particles dispersed in the synapse most likely represent hemi-channels produced by the dissasembly of channels previously arranged in plaques.

Gap junctions in the prothoracic glands of the tobacco hornworm, Manduca sexta

1992 ◽  
Vol 50 (1) ◽  
pp. 706-707
Author(s):  
Ji-da Dai ◽  
M. Joseph Costello ◽  
Lawrence I. Gilbert
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
Manduca Sexta ◽  
Freeze Fracture ◽  
Cell Communication ◽  
Cellular Level ◽  
Thin Sections ◽  
Prothoracic Glands ◽  
Polyhydroxylated Steroids ◽  
Stimulation Of

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.

Freeze-fracture studies of frog atrial fibres

1976 ◽  
Vol 22 (2) ◽  
pp. 427-434
Author(s):  
F. Mazet ◽  
J. Cartaud
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Outer Membrane ◽  
Electrical Coupling ◽  
Freeze Fracture ◽  
Present Knowledge ◽  
Inner Membrane ◽  
Freeze Fracturing ◽  
Morphological Variant ◽  
Characteristic Features

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the “gap junction” or “nexus”. The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.

scholarly journals Gap junction structures after experimental alteration of junctional channel conductance.

1985 ◽  
Vol 101 (5) ◽  
pp. 1741-1748 ◽  
Author(s):  
T M Miller ◽  
D A Goodenough
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Electron Micrographs ◽  
Time Course ◽  
Structural Changes ◽  
Lucifer Yellow ◽  
Freeze Fracture ◽  
Channel Conductance ◽  
Experimental Alteration ◽  
Quick Freezing

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.

Heart gap junction preparations reveal hemiplaques by atomic force microscopy

1995 ◽  
Vol 268 (4) ◽  
pp. C968-C977 ◽  
Author(s):  
R. Lal ◽  
S. A. John ◽  
D. W. Laird ◽  
M. F. Arnsdorf
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Long Range ◽  
Connexin 43 ◽  
Plasma Membranes ◽  
Aqueous Media ◽  
Freeze Fracture ◽  
Isolated Rat Heart ◽  
Hexagonal Packing ◽  
Atomic Force

Current structural models of gap junctions indicate two apposed plasma membranes with hexagonally packed hemichannels in each membrane aligning end to end. These channels connect the cytoplasms of contacting cells. Images of isolated rat heart gap junctions have been made with the atomic force microscope in aqueous media. We show that native cardiac gap junctions have a thickness of 25 +/- 0.6 nm. This decreases to 17 nm when they are treated with trypsin, which is known to remove some cytoplasmic components of connexin 43. Imaging shows subunits with a center to center spacing of approximately 9-10 nm and long range hexagonal packing, measurements in agreement with studies using freeze-fracture and negative-stain electron microscopy. In addition to gap junctions, we imaged structures that had all the characteristics of native gap junctions except their thickness was limited to 9-11 nm. They also show long range hexagonal packing and center to center spacing of 9-10 nm. These structures decrease in thickness, to 6-9 nm, when treated with trypsin. We have called these structures hemiplaques. They appear to be present endogenously in the preparation, as we have ruled out their being an artifact of imaging by AFM. However, it remains to be determined if they are a consequence of the procedure used in isolating gap junctions or a possible intermediary in gap junction formation.

scholarly journals Ultrastructural and molecular characteristics of crayfish photoreceptor membranes.

1976 ◽  
Vol 69 (3) ◽  
pp. 721-732 ◽  
Author(s):  
H R Fernandez ◽  
E E Nickel
Keyword(s):  
Freeze Fracture ◽  
Photoreceptor Cells ◽  
Molecular Characteristics ◽  
Thin Sections ◽  
Freeze Fracturing ◽  
Mean Diameter ◽  
Hexagonal Arrangement ◽  
Photoreceptor Membranes

The ultrastructure of photoreceptor cells of the crayfish (P. clarkii) has been examined by means of thin sections and freeze-fracturing. The study reveals that in the photoreceptor membranes there are particles associated primarily with the A faces of freeze-fracture preparations which have a mean diameter of 80-84 A and a density of 6,600 per per micrometer2. Treatment of the retina with digitonin (a substance capable of extracting visual photopigments) in Ringer's causes marked disruption of the hexagonal arrangement of the microvilli, breakdown of the microvilli into smaller segments, and gradual removal of the particles. The estimated photopigment concentration in the microvillus is 4,000 per micrometer. It is suggested that the observed particles represent the photopigment in situ.

Differences between cytoplasmic surfaces of deep-etched heart and liver gap junctions

1985 ◽  
Vol 249 (3) ◽  
pp. H690-H693 ◽  
Author(s):  
Y. Shibata ◽  
C. K. Manjunath ◽  
E. Page
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Published Data ◽  
Rapid Freezing ◽  
Protein Subunit ◽  
Rat Hearts ◽  
Gap Junctional ◽  
Isolated Liver ◽  
Junction Proteins

We have compared the ultrastructures of the cytoplasmic surfaces (CS) of isolated, glutaraldehyde-fixed gap junctional pellets from rat ventricles and liver by rapid freezing on a liquid helium-cooled surface, freeze fracture, deep etching, and double-axis rotary replication (J. Microsc. Oxford 137: 121-123, 1984). Deep-etched unproteolyzed cardiac junctions [protein subunit relative molecular wt (Mr) 44,000-47,000], isolated with phenylmethylsulfonylfluoride (PMSF) [Am. J. Physiol. 246 (Heart Circ. Physiol. 15): H865-H875, 1984; C.K. Manjunath, G.E. Goings, and E. Page. Proteolysis of cardiac gap junctions during their isolation from rat hearts. J. Membr. Biol. In press.] had particulate CS, while proteolyzed cardiac junctions (subunit Mr 29,500) made without PMSF and liver junctions (Mr 28,000) made with or without PMSF had nonparticulate CS. Taken together with our previous findings that electron micrographs of thin-sectioned isolated unproteolyzed cardiac junctions have urea-resistant fuzzy CS coatings [Am. J. Physiol. 246 (Heart Circ. Physiol. 15): H865-H875, 1984], that proteolyzed cardiac junctions and isolated liver junctions lack this fuzzy layer, and that the CS of in situ cardiac junctions is fuzzy [J. Membr. Biol. 78: 147-155, 1984; Am. J. Physiol. 246 (Heart Circ. Physiol 15): H865-H875, 1984], and published data that in situ freeze-etched liver gap junctions are nonparticulate (Cell 30: 395-406, 1982), these new observations strongly indicate that CS components of heart and liver gap junction proteins are structurally different.

Gap junction associated filaments in testis interstitial cells of the rat

1978 ◽  
Vol 36 (2) ◽  
pp. 550-551
Author(s):  
J.F. David-Ferreira ◽  
K.L. David-Ferreira ◽  
M.H. Miranda ◽  
J.C. Lemos
Keyword(s):  
Gap Junction ◽  
Freeze Fracture ◽  
Interstitial Cells ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Thin Sections ◽  
Cacodylate Buffer ◽  
Old Rats ◽  
The Relationship ◽  
Made In

The testis interstitial cells of the rat are closely associated forming small aggregates in the vicinity of the vessels. The finality of the present study is to analise the relationship between the interstitial cells and to describe some particularities of their junctions.The observations were made in thin sections from testis of 2 to 3 months old rats fixed by immersion or perfusion with 2 to 3% glutaral- dehyde in s-collidine or cacodylate buffer followed by 2% osmium tetro-xyde in the same buffers. Some specimens have been prepared following the technique of Shea. The thin sections obtained after embedding in Epon were double stained in uranyl acetate and lead citrate. For the cryofracture study the tissues were fixed in 3% glutaraldehyde in cacodylate buffer, impregnated in 28% glycerol, frozen in Freon 22 and stored in liquid nitrogen. Freeze fracture and platinum-carbon shadowing were done in a Balzers 300.

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