Quantitative Freeze Fracture Studies of Gap Junctions in Somatic Cell Hybrids, Their Parents, and Revertants

1976 ◽  
Vol 34 ◽  
pp. 218-219
Author(s):  
W. J. Larsen ◽  
R. Azarnia ◽  
W. R. Loewenstein
Keyword(s):  
Gap Junctions ◽  
Striated Muscle ◽  
Freeze Fracture ◽  
Cell Movement ◽  
Dye Molecules ◽  
Somatic Cell Hybrids ◽  
Cell Coupling ◽  
Normal Cells ◽  
Mediate Cell ◽  
Almost All

Although the physiological significance of the gap junction remains unspecified, these membrane specializations are now recognized as common to almost all normal cells (excluding adult striated muscle and some nerve cells) and are found in organisms ranging from the coelenterates to man. Since it appears likely that these structures mediate the cell-to-cell movement of ions and small dye molecules in some electrical tissues, we undertook this study with the objective of determining whether gap junctions in inexcitable tissues also mediate cell-to-cell coupling.To test this hypothesis, a coupling, human Lesh-Nyhan (LN) cell was fused with a non-coupling, mouse cl-1D cell, and the hybrids, revertants, and parental cells were analysed for coupling with respect both to ions and fluorescein and for membrane junctions with the freeze fracture technique.

Calmodulin-mediated gating of lens gap junction channels in vesicles

1984 ◽  
Vol 42 ◽  
pp. 134-137
Author(s):  
Camillo Peracchia ◽  
Stephen J. Girsch
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Orthogonal Arrays ◽  
Eye Lens ◽  
Lens Fiber ◽  
Cell Coupling ◽  
Particle Spacing ◽  
Fiber Cells ◽  
Gap Junction Channels ◽  
Communicating Junctions

The fiber cells of eye lens communicate directly with each other by exchanging ions, dyes and metabolites. In most tissues this type of communication (cell coupling) is mediated by gap junctions. In the lens, the fiber cells are extensively interconnected by junctions. However, lens junctions, although morphologically similar to gap junctions, differ from them in a number of structural, biochemical and immunological features. Like gap junctions, lens junctions are regions of close cell-to-cell apposition. Unlike gap junctions, however, the extracellular gap is apparently absent in lens junctions, such that their thickness is approximately 2 nm smaller than that of typical gap junctions (Fig. 1,c). In freeze-fracture replicas, the particles of control lens junctions are more loosely packed than those of typical gap junctions (Fig. 1,a) and crystallize, when exposed to uncoupling agents such as Ca++, or H+, into pseudo-hexagonal, rhombic (Fig. 1,b) and orthogonal arrays with a particle-to-particle spacing of 6.5 nm. Because of these differences, questions have been raised about the interpretation of the lens junctions as communicating junctions, in spite of the fact that they are the only junctions interlinking lens fiber cells.

MP26 is a lens junctional protein: Analysis with monoclonal antibodies

1984 ◽  
Vol 42 ◽  
pp. 118-121
Author(s):  
Ross Johnson ◽  
Jane Sas ◽  
Catherine Nelson ◽  
Sue Schik ◽  
Brad Quade ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Cell Movement ◽  
Protein Analysis ◽  
Cell Junctions ◽  
Major Protein ◽  
Definitive Answer ◽  
Junction Protein ◽  
Junctional Protein

Investigators with interests in cell junctions, and gap junctions in particular, have been intrigued over the last 5-10 years by the extensive junctional specializations found in the lens. Attention has focused on the suggested role of MP26 as the major protein component. The fundamental question has been whether the abundant junctions seen with thin-section or freeze-fracture analyses correspond to lens “gap junctions.” That is, do these cell surface specializations between lens fiber cells provide for the direct cell-to-cell movement of small molecules? Unfortunately, we still lack a definitive answer to the question. For some investigators, enthusiasm waned when protein analysis uncovered no homology between MP26 and a major liver gap junction protein. However, we have much to learn about the nature and role of the lens junction.

Light and Electron Microscopic Studies Regarding Cell Contractility and Cell Coupling in Light Sensitive Smooth Muscle Cells from the Isolated Frog Iris Sphincter

1987 ◽  
Vol 42 (7-8) ◽  
pp. 977-985 ◽  
Author(s):  
Klaus V. Wolf
Keyword(s):  
Gap Junctions ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Sphincter Muscle ◽  
Cell Coupling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Cell Contractility ◽  
Microscopic Studies ◽  
Iris Sphincter

(1) In light microscopical studies of living isolated frog irises, it was found that the maximal areas of experimentally light induced contractions in the m. sphincter pupillae were located beneath small illuminated regions. There were no visible contractions of muscle cells outside the illuminated areas. It was shown that exposure to light could directly cause contractions of isolated single sphincter muscle cells. (2) Junctional structures of the iris sphincter cells were studied by means of thin sections and freeze fracture electron microscopy. Intermediate junctions, a few focal tight junctions and occasional small gap junctions were identified. Pit containing intramembranous particles which resemble gap junction connexons were found in large numbers, dispersed over the plasmalemmas of sphincter muscle cells. From these physiological and morphological observations, it is concluded that sphincter muscle cells of the frog iris may be coupled via gap junctions, but that the cell coupling is not sufficiently extensive to form the basis for a functional syncytium.

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.

scholarly journals Growth factor dependent changes in nanoscale architecture of focal adhesions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karin Legerstee ◽  
Tsion E. Abraham ◽  
Wiggert A. van Cappellen ◽  
Alex L. Nigg ◽  
Johan A. Slotman ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Intermediate Layer ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Bottom Layer ◽  
Vertical Axis ◽  
Longitudinal Distribution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Mediate Cell

AbstractFocal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement.

scholarly journals Intercellular calcium signalling between chondrocytes and synovial cells in co-culture

1998 ◽  
Vol 329 (3) ◽  
pp. 681-687 ◽  
Author(s):  
Paola D'ANDREA ◽  
Alessandra CALABRESE ◽  
Micaela GRANDOLFO
Keyword(s):  
Gap Junctions ◽  
Intercellular Communication ◽  
Structural Changes ◽  
Articular Chondrocytes ◽  
Cell Metabolism ◽  
Second Messengers ◽  
Calcium Signalling ◽  
Cell Types ◽  
Synovial Cells ◽  
Cell Coupling

Intercellular communication allows the co-ordination of cell metabolism between tissues as well as sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks that favour the intercellular exchange of nutrients and second messengers. Heterologous intercellular communication was studied in co-cultures of articular chondrocytes and HIG-82 synovial cells by measuring mechanically induced cytosolic changes in Ca2+ ion levels by digital fluorescence video imaging. In confluent co-cultures, mechanical stimulation induced intercellular Ca2+ waves that propagated to both cell types with similar kinetics. Intercellular wave spreading was inhibited by 18α-glycyrrhetinic acid and by treatments inhibiting the activation of purinoreceptors, suggesting that intercellular signalling between these two cell types occurs both through gap junctions and ATP-mediated paracrine stimulation. In rheumatoid arthritis the formation of the synovial pannus induces structural changes at the chondrosynovial junction, where chondrocyte and synovial cells come into close apposition: these results provide the first evidence for direct intercellular communication between these two cell types.

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.

Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate

2000 ◽  
Vol 113 (8) ◽  
pp. 1365-1372 ◽  
Author(s):  
H. Niessen ◽  
H. Harz ◽  
P. Bedner ◽  
K. Kramer ◽  
K. Willecke
Keyword(s):  
Gap Junctions ◽  
Hela Cells ◽  
Second Messenger ◽  
Pancreatic Acinar Cells ◽  
Cell Coupling ◽  
Liver Parenchymal ◽  
Fura 2 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Messenger Molecules ◽  
Parenchymal Cells

Intercellular propagation of signals through connexin32-containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin-transfected HeLa cells was standardized by measuring the spreading of microinjected Mn(2+) that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca(2+) wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32-containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.

Ultrastructural analysis of the apical ectodermal ridge during vertebrate limb morphogenesis

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 223-232
Author(s):  
John F. Fallon ◽  
Robert O. Kelley
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Freeze Fracture ◽  
Apical Ectodermal Ridge ◽  
Structural Requirements ◽  
Limb Morphogenesis ◽  
Vertebrate Limb ◽  
The Difference ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron

The fine structure of the apical ectodermal ridge of five phylogenetically divergent orders of mammals and two orders of birds was examined using transmission and freeze fracture electron microscopy. Numerous large gap junctions were found in all apical ectodermal ridges studied. This was in contrast to the dorsal and ventral limb ectoderms where gap junctions were always very small and sparsely distributed. Thus, gap junctions distinguish the inductively active apical epithelium from the adjacent dorsal and ventral ectoderms. The distribution of gap junctions in the ridge was different between birds and mammals but characteristic within the two classes. Birds, with a pseudostratified columnar apical ridge, had the heaviest concentration of gap junctions at the base of each ridge cell close to the point where contact was made with the basal lamina. Whereas mammals, with a stratified cuboidal to squamous apical ridge, had a more uniform distribution of gap junctions throughout the apical epithelium. The difference in distribution for each class may reflect structural requirements for coupling of cells in the entire ridge. We propose that all cells of the apical ridges of birds and mammals are electrotonically and/or metabolically coupled and that this may be a requirement for the integrated function of the ridge during limb morphogenesis.

A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling

1992 ◽  
Vol 101 (3) ◽  
pp. 503-508
Author(s):  
R. Newman ◽  
G.W. Butcher ◽  
B. Bullard ◽  
K.R. Leonard
Keyword(s):  
Gold Particle ◽  
Colloidal Gold ◽  
Striated Muscle ◽  
Flight Muscle ◽  
Insect Flight ◽  
Insect Flight Muscle ◽  
Fab Fragment ◽  
Thin Filaments ◽  
Gold Particles ◽  
Almost All

Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosin-troponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately +/− 4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.

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