Dissociation of lens fibre gap junctions releases MP70

1988 ◽  
Vol 91 (3) ◽  
pp. 415-421 ◽  
Author(s):  
J. Kistler ◽  
S. Bullivant
Keyword(s):  
Electron Microscope ◽  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Membrane Structures ◽  
Treatment Results ◽  
Double Membrane ◽  
Lens Fibre ◽  
Junction Proteins

MIP and MP70 are putative gap junction components in the plasma membranes of the mammalian lens fibre cells. We show now that MP70 can be solubilized separately from MIP in mild detergent solutions, and that this treatment results in the dissociation of the fibre gap junctions. Solubilized MP70 was isolated as 16.9 S particles by velocity gradient centrifugation and in the electron microscope had the appearance of short double-membrane structures consistent with connexon-pairs. These observations open a new experimental avenue in which to characterize separately the two putative lens gap junction proteins structurally and functionally.

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 Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions

2002 ◽  
Vol 365 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Translation System ◽  
Alternative Mechanism ◽  
Protein Cleavage ◽  
Membrane Components ◽  
Gap Junction Hemichannels

Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.

scholarly journals On gap junction structure.

1980 ◽  
Vol 86 (1) ◽  
pp. 190-198 ◽  
Author(s):  
G Zampighi ◽  
J M Corless ◽  
J D Robertson
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rat Liver ◽  
Extracellular Space ◽  
Plasma Membranes ◽  
Negative Staining ◽  
The Other ◽  
Central Axis ◽  
Thin Sectioning ◽  
Junction Structure

We have studied the stain distribution within rat liver gap junctions for specimens prepared by thin sectioning and negative staining. Pools of stain molecules exist in two specific locations with respect to the distinctive morphological units (connexons) of the junction. One pool of stain surrounds the connexons and is restricted to the extracellular space in the gap between the adjacent plasma membranes. The other pool of stain is located along in the central axis of each connexon, measures 1-2 nm in diameter and 4-5 nm in length, and is restricted to the gap region. On rare occasions, barely discernible linear densities seem to extend from this latter pool of stain and traverse the entire width of the junction. The data indicate the existence of a hydrophilic cavity along the central axis of te connexon which, in most instances, is restricted to the gap region. However, the precise depth to which this cavity may further extend along the connexon axis is still uncertain.

Synthesis and assembly of human beta 1 gap junctions in BHK cells by DNA transfection with the human beta 1 cDNA

1995 ◽  
Vol 108 (12) ◽  
pp. 3725-3734 ◽  
Author(s):  
N.M. Kumar ◽  
D.S. Friend ◽  
N.B. Gilula
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Freeze Fracture ◽  
Growth Control ◽  
Double Membrane ◽  
Correct Orientation ◽  
Junctional Communication ◽  
Junction Protein ◽  
Particle Aggregates ◽  
Metallothionein Promoter

Gap junctional communication is important in many physiological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human beta 1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional beta 1 connexin was detected only in cells transfected with beta 1 connexin cDNA in the correct orientation (beta 1-BHK). Analysis of the cells by light microscopic immunocytochemistry indicated that beta 1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomerization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.

A non-connexon protein (MIP) is involved in eye lens gap-junction formation

1989 ◽  
Vol 93 (3) ◽  
pp. 509-513
Author(s):  
W.T. Gruijters
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Eye Lens ◽  
Specific Interactions ◽  
Domain Specific ◽  
Lens Fibre ◽  
Junction Formation ◽  
New Perspective

New immunolocalization data put the role of the lens MP26 (MIP) protein in a new perspective. During maturation of lens fibre cells, MIP is found to associate specifically with two structures, gap junctions and cell interlocking processes (known as ball and socket domains). It is significant that the zone in which these associations are most striking is discrete, coinciding with the zone of rapidly enlarging junctional plaques and newly forming ball and socket domains. Observation of domain-specific interactions of MIP with forming gap junctions and ball and socket domains suggests that MIP may be involved in the formation of close membrane appositions. Furthermore, previous ambiguities in the literature over the presence of MIP in gap junctions are clarified by the knowledge that, in situ, MIP associates strongly with gap junctions for only a brief period (with less than about 5% of all lens gap junctions at any one time) during the assembly of junctional plaques.

scholarly journals Visualization of Annular Gap Junction Vesicle Processing: The Interplay between Annular Gap Junctions and Mitochondria

2018 ◽  
Author(s):  
Cheryl L. Bell ◽  
Teresa I. Shakespeare ◽  
Sandra A. Murray
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cell Communication ◽  
Super Resolution ◽  
Protective Role ◽  
Annular Gap ◽  
Transmission Electron ◽  
Junction Proteins

It is becoming clear that in addition to gap junctions, playing a role in cell-cell communication, gap junction proteins, connexins, located in cytoplasmic-compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unknown. In this study, immunocytochemical, super-resolution and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structure and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures, annular gap junctions, were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 deliver to the mitochondria. Furthermore, it points to the need for investigating trafficking of Cx43 to cytoplasmic compartments and annular gap junction as more than only a vesicle destined for degradation.

scholarly journals βPS-Integrin acts downstream of Innexin 2 in modulating stretched cell morphogenesis in the Drosophila ovary

2021 ◽  
Author(s):  
Yi-Chia Huang ◽  
Kuan-Han Chen ◽  
Yu-Yang Chen ◽  
Liang-Hsuan Tsao ◽  
Tsung-Han Yeh ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Adaptor Proteins ◽  
Follicle Cells ◽  
Cell Morphogenesis ◽  
Independent Manner ◽  
Gap Junction Proteins ◽  
Intercellular Exchange ◽  
Drosophila Ovary ◽  
Junction Proteins

Abstract During oogenesis, a group of specialized follicle cells, known as stretched cells, flatten drastically from cuboidal to squamous shape. While morphogenesis of epithelia is critical for organogenesis, genes and signaling pathways involved in this process remain to be revealed. In addition to formation of gap junctions for intercellular exchange of small molecules, gap junction proteins form channels or act as adaptor proteins to regulate various cellular behaviors. In invertebrates, gap junction proteins are Innexins. Knockdown of Innexin 2 but not other Innexins expressed in follicle cells attenuates stretched cell morphogenesis. Interestingly, blocking of gap junctions with an inhibitor carbenoxolone does not affect stretched cell morphogenesis, suggesting that Innexin 2 might control stretched cell flattening in a gap-junction-independent manner. An excessive level of βPS-Integrin encoded by myospheroid is detected in Innexin 2 mutant cells specifically during stretched cell morphogenesis. Simultaneous knockdown of Innexin 2 and myospheroid partially rescues the morphogenetic defect resulted from Innexin 2 knockdown. Furthermore, reduction of βPS-Integrin is sufficient to induce early stretched cell flattening. Taken together, our data suggest that βPS-Integrin acts downstream of Innexin 2 in modulating stretched cell morphogenesis.

scholarly journals COEXISTENCE OF GAP AND SEPTATE JUNCTIONS IN AN INVERTEBRATE EPITHELIUM

1971 ◽  
Vol 50 (1) ◽  
pp. 92-101 ◽  
Author(s):  
A. J. Hudspeth ◽  
J. P. Revel
Keyword(s):  
Electron Microscope ◽  
Gap Junctions ◽  
Gap Junction ◽  
Extracellular Space ◽  
Intercellular Junctions ◽  
Septate Junction ◽  
High Electron ◽  
Septate Junctions ◽  
Electrotonic Coupling ◽  
Lanthanum Tracer

The intercellular junctions of the epithelium lining the hepatic caecum of Daphnia were examined. Electron microscope investigations involved both conventionally fixed material and tissue exposed to a lanthanum tracer of the extracellular space. Both septate junctions and gap junctions occur between the cells studied. The septate junctions lie apically and resemble those commonly discerned between cells of other invertebrates. They are atypical in that the high electron opacity of the extracellular space obscures septa in routine preparations. The gap junctions are characterized by a uniform 30 A space between apposed cell membranes. Lanthanum treatment of gap junctions reveals an array of particles of 95 A diameter and 120 A separation lying in the plane of the junction. As this pattern closely resembles that described previously in vertebrates, it appears that the gap junction is phylogenetically widespread. In view of evidence that the gap junction mediates intercellular electrotonic coupling, the assignment of a coupling role to other junctions, notably the septate junction, must be questioned wherever these junctions coexist.

scholarly journals Visualization of Annular Gap Junction Vesicle Processing: The Interplay Between Annular Gap Junctions and Mitochondria

2018 ◽  
Vol 20 (1) ◽  
pp. 44 ◽  
Author(s):  
Cheryl Bell ◽  
Teresa Shakespeare ◽  
Amber Smith ◽  
Sandra Murray
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cell Communication ◽  
Super Resolution ◽  
Protective Role ◽  
Annular Gap ◽  
Transmission Electron ◽  
Junction Proteins

It is becoming clear that in addition to gap junctions playing a role in cell–cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures—annular gap junctions—were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.

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