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.

scholarly journals Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly.

1994 ◽  
Vol 127 (6) ◽  
pp. 1895-1905 ◽  
Author(s):  
P D Lampe
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Freeze Fracture ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Junctional Communication ◽  
Junction Protein

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.

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.

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.

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.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

Quick-Freezing to Catch the Membrane Changes that Occur During Exocytosis

1977 ◽  
Vol 35 ◽  
pp. 676-679
Author(s):  
J.E. Heuser
Keyword(s):  
Synaptic Vesicle ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Frog Neuromuscular Junction ◽  
The Past ◽  
Synaptic Vesicle Exocytosis ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Membrane Changes

The technique that we have used to capture synaptic vesicle exocytosis at the frog neuromuscular junction - that of quick-freezing muscles followed by freeze fracture (3) or freeze substitution (6) - works sufficiently well now that it may be useful in other sorts of membrane studies, or studies of fast structural changes with the electron microscope. This note reviews the quickfreezing technique we use, and describes its application to the problem of synaptic vesicle exocytosis and recycling at the synapse.Here, many of the membrane changes of interest occur during the brief delay in synaptic transmission, on a time scale of milliseconds or fractions of milliseconds, and leave only traces thereafter. In the past, we have studied these left-over traces in tissues fixed with the standard chemicals for electron microscopy (1), and have inferred from them that vesicles discharge the quanta of neurotransmitters, as the physiologists would predict.

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.

Intercellular communication between epithelial and fiber cells of the eye lens

1994 ◽  
Vol 107 (4) ◽  
pp. 799-811 ◽  
Author(s):  
S. Bassnett ◽  
J.R. Kuszak ◽  
L. Reinisch ◽  
H.G. Brown ◽  
D.C. Beebe
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Cell Types ◽  
Lens Epithelium ◽  
Fiber Cell ◽  
Fracture Plane ◽  
Metabolic Cooperation ◽  
Fiber Cells ◽  
Resistance Pathway

Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit ‘metabolic cooperation’ between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4 degrees C or by treatment with octanol and which excluded large (approximately 10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.

Increased gap junction assembly between cultured cells upon cholesterol supplementation

1990 ◽  
Vol 96 (2) ◽  
pp. 231-238
Author(s):  
R. Meyer ◽  
B. Malewicz ◽  
W.J. Baumann ◽  
R.G. Johnson
Keyword(s):  
Gap Junction ◽  
Actinomycin D ◽  
Cultured Cells ◽  
Lucifer Yellow ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Dye Transfer ◽  
Novikoff Hepatoma ◽  
Fourfold Increase ◽  
Cholesterol Supplementation

Novikoff hepatoma cells provide an excellent model system for the study of gap junction assembly, a process that could be influenced by lipids and other factors at numerous points. Since it is possible to alter the cellular levels of cholesterol in these cells, it was added to the cells in serum-supplemented medium and changes in gap junction assembly were evaluated. Cells were dissociated and reaggregated following exposure to a range of cholesterol concentrations for 24 h. A five- to sixfold increase in the number of aggregated gap junction particles and a 50% increase in cellular cholesterol content were observed with 20 microM added cholesterol. A 1-h exposure to added cholesterol, during cell reaggregation, resulted in a fourfold increase in the number of aggregated gap junction particles, demonstrating that the effect was rapid. The number of aggregated gap junction particles and formation plaque areas were used as measures of junction assembly and assayed by quantitative freeze-fracture and electron microscopy. Junctional permeabilities were evaluated by means of dye transfer times following the intracellular microinjection of Lucifer Yellow. Increased dye transfer was observed between cholesterol-treated cells, which suggested that the increase in assembly was accompanied by an increase in junction permeability. Cells were treated with cycloheximide (100 micrograms ml-1) and actinomycin D (10 micrograms ml-1) to determine whether protein and RNA syntheses were involved in the enhanced gap junction assembly. Cycloheximide but not actinomycin D blocked the increased junction assembly observed with added cholesterol. These results suggested that protein synthesis, but not RNA synthesis, is necessary for the increased gap junction formation observed.(ABSTRACT TRUNCATED AT 250 WORDS)

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