scholarly journals Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

2020 ◽  
pp. jcs.252726
Author(s):  
Rachael P. Norris ◽  
Mark Terasaki
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
New Technology ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Annular Gap

Gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. Here, we systematically investigated the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume corresponding to ∼35 cells, every labeled structure was categorized and its surface area was measured. Measurements support the concept that multiple connexosomes form from larger invaginated gap junctions. Subsequently, the inner and outer membranes separate, Cx43 immunogenicity is lost from the outer membrane, and the inner membrane appears to undergo fission. One pathway for processing involves lysosomes, based on localization of Cathespin B to some processed connexosomes. In summary, this study demonstrates new technology for high-resolution analyses of gap junction processing.

scholarly journals Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

2020 ◽  
Author(s):  
Rachael P. Norris ◽  
Mark Terasaki
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Annular Gap ◽  
Cell Cell

AbstractGap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. Here, we systematically studied the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume of electron micrographs, every labeled structure was categorized and counted. Surface area measurements indicate that large connexosomes undergo fission. Subsequent modifications are separation of inner and outer membranes, loss of Cx43 from the outer membrane, and outward budding of the modified membranes. We also documented several clear examples of organelle transfer from one cell to another by gap junction internalization. We discuss how connexosome formation and processing may be a novel means for gap junctions to mediate cell-cell communication.

scholarly journals Connexin 43 K63-polyubiquitination on lysines 264 and 303 regulates gap junction internalization

2017 ◽  
Author(s):  
Rachael M. Kells-Andrews ◽  
Rachel A. Margraf ◽  
Charles G. Fisher ◽  
Matthias M. Falk
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Room Temperature ◽  
Cell Communication ◽  
Mapk Phosphorylation ◽  
Annular Gap ◽  
Summary Statement ◽  
Triton X

ABSTRACTGap junctions (GJs) assembled from connexin (Cx) proteins play a pivotal role in cell-to-cell communication by forming channels that connect the cytosols of adjacent cells. Connexin 43, the best-studied Cx, is ubiquitously expressed in vertebrates. While phosphorylation is known to regulate multiple aspects of GJ function, much less is known about the role ubiquitination plays in these processes. Here we show by using ubiquitination-type specific antibodies and Cx43 lysine (K) to arginine (R) mutants that a portion of Cx43 in GJs can become K63-polyubiquitinated on K264 and K303. Relevant Cx43 K/R mutants assembled significantly larger GJ plaques, exhibited much longer protein half-lives and were internalization impaired. Interestingly, ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitination may be triggered by phosphorylation. Phospho-specific Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282, and 255, well-known regulatory PKC and MAPK phosphorylation sites. Together, these novel findings suggest that upon internalization, some Cx43 in GJs becomes K63-polyubiquitinated, ubiquitination is critical for GJ internalization, and that K63-polyubiquitination may be induced by Cx phosphorylation.Summary StatementHere we show that connexin 43 in gap junctions becomes K63-poly ubiquitinated on lysines 264 and 303 and its requirement for gap junction endocytosis. These novel findings significantly contribute to our understanding of GJ turnover and patho-/physiology.Abbreviations usedAGJannular gap junctionAMSHassociated molecule with the SH3 domain of STAMCMEclathrin-mediated endocytosisCxConnexinCx43Connexin 43DUBdeubiquitinaseGJgap junctionMonoUbmonoubiquitinNedd4-1neural precursor cell expressed developmentally down-regulated protein 4-1PMplasma membranePolyUbpolyubiquitinTPA12-O-Tetradecanoylphorbol 13-AcetateTX-100Triton X-100RTroom temperatureUbubiquitin

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 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.

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 509-522
Author(s):  
R. Minkoff ◽  
S.B. Parker ◽  
E.L. Hertzberg
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Uniform Distribution ◽  
Rat Liver ◽  
Connexin 43 ◽  
Cell Communication ◽  
Exterior Surface ◽  
Junction Protein ◽  
Primary Palate ◽  
Gap Junction Protein

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 × 10(3) Mr, connexin 32) and heart (43 × 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 × 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the ‘heart’ 43 × 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

scholarly journals Fibroblast Growth Factor 4 Directs Gap Junction Expression in the Mesenchyme of the Vertebrate Limb Bud

1997 ◽  
Vol 138 (5) ◽  
pp. 1125-1137 ◽  
Author(s):  
H. Makarenkova ◽  
D.L. Becker ◽  
C. Tickle ◽  
A.E. Warner
Keyword(s):  
Growth Factor ◽  
Gap Junctions ◽  
Gap Junction ◽  
Fibroblast Growth Factor ◽  
Connexin 43 ◽  
Limb Bud ◽  
Functional Coupling ◽  
Fibroblast Growth ◽  
Mesenchyme Cells

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell–cell communication and polarizing signaling are intimately connected.

Polyamines regulate gap junction communication in connexin 43-expressing cells

2001 ◽  
Vol 357 (2) ◽  
pp. 489-495 ◽  
Author(s):  
Leonard SHORE ◽  
Pauline McLEAN ◽  
Susan K. GILMOUR ◽  
Malcolm B. HODGINS ◽  
Malcolm E. FINBOW
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ornithine Decarboxylase ◽  
Normal Tissue ◽  
Connexin 43 ◽  
Cell Communication ◽  
Cell Types ◽  
Decarboxylase Activity ◽  
Gap Junction Communication ◽  
Different Cell Types

The control of cell–cell communication through gap junctions is thought to be crucial in normal tissue function and during various stages of tumorigenesis. However, few natural regulators of gap junctions have been found. We show here that increasing the activity of ornithine decarboxylase, or adding polyamines to the outside of cells, increases the level of gap junction communication between various epithelial cells. Conversely, reduction of ornithine decarboxylase activity decreases the level of gap junction communication. This regulation is dependent upon the expression of connexin 43 (Cx43 or Cxα1), which is a major connexin expressed in many different cell types, and involves an increase in Cx43 and its cellular re-distribution.

scholarly journals Heteromerization of Innexin Gap Junction Proteins Regulates Epithelial Tissue Organization in Drosophila

2006 ◽  
Vol 17 (4) ◽  
pp. 1676-1685 ◽  
Author(s):  
Corinna Lehmann ◽  
Hildegard Lechner ◽  
Birgit Löer ◽  
Martin Knieps ◽  
Sonja Herrmann ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ectopic Expression ◽  
Cell Communication ◽  
Epithelial Tissue ◽  
Surface Plasmon Resonance Analysis ◽  
Gap Junction Channels ◽  
Tissue Organization ◽  
Biochemical Fractionation

Gap junctions consist of clusters of intercellular channels, which enable direct cell-to-cell communication and adhesion in animals. Whereas deuterostomes, including all vertebrates, use members of the connexin and pannexin multiprotein families to assemble gap junction channels, protostomes such as Drosophila and Caenorhabditis elegans use members of the innexin protein family. The molecular composition of innexin-containing gap junctions and the functional significance of innexin oligomerization for development are largely unknown. Here, we report that heteromerization of Drosophila innexins 2 and 3 is crucial for epithelial organization and polarity of the embryonic epidermis. Both innexins colocalize in epithelial cell membranes. Innexin3 is mislocalized to the cytoplasm in innexin2 mutants and is recruited into ectopic expression domains defined by innexin2 misexpression. Conversely, RNA interference (RNAi) knockdown of innexin3 causes mislocalization of innexin2 and of DE-cadherin, causing cell polarity defects in the epidermis. Biochemical interaction studies, surface plasmon resonance analysis, transgenesis, and biochemical fractionation experiments demonstrate that both innexins interact via their C-terminal cytoplasmic domains during the assembly of heteromeric channels. Our data provide the first molecular and functional demonstration that innexin heteromerization occurs in vivo and reveal insight into a molecular mechanism by which innexins may oligomerize into heteromeric gap junction channels.

scholarly journals Primary Cultures of Chick Osteocytes Retain Functional Gap Junctions between Osteocytes and between Osteocytes and Osteoblasts

2007 ◽  
Vol 13 (2) ◽  
pp. 108-117 ◽  
Author(s):  
Hiroshi Kamioka ◽  
Yoshihito Ishihara ◽  
Hans Ris ◽  
Sakhr A. Murshid ◽  
Yasuyo Sugawara ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Lucifer Yellow ◽  
Primary Cultures ◽  
Bone Matrix ◽  
Dye Coupling ◽  
Direct Demonstration

The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18α-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.

scholarly journals Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

2021 ◽  
pp. mbc.E20-12-0797
Author(s):  
Caitlin Hyland ◽  
Michael Mfarej ◽  
Giorgos Hiotis ◽  
Sabrina Lancaster ◽  
Noelle Novak ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cardiac Tissue ◽  
Cultured Cells ◽  
Cell Communication ◽  
Cardiovascular Development ◽  
Zebrafish Model ◽  
Cx43 Protein ◽  
Molecular Events

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of connexins of only 1-5 hours, resulting in constant endocytosis and biosynthetic replacement of gap junction channels has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43∆256-289) and a zebrafish model ( cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx 43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that Cx43 gap junction endocytosis is an essential aspect of gap junction function and when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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