scholarly journals Zonula Occludens-1 Alters Connexin43 Gap Junction Size and Organization by Influencing Channel Accretion

2005 ◽  
Vol 16 (12) ◽  
pp. 5686-5698 ◽  
Author(s):  
Andrew W. Hunter ◽  
Ralph J. Barker ◽  
Ching Zhu ◽  
Robert G. Gourdie
Keyword(s):  
Image Analysis ◽  
Gap Junction ◽  
Peptide Inhibitor ◽  
Proper Function ◽  
Plaque Size ◽  
Confocal Image ◽  
Zonula Occludens ◽  
Genetic Tagging ◽  
Zonula Occludens 1 ◽  
Gap Junctional

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.

scholarly journals Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover

2017 ◽  
Vol 28 (25) ◽  
pp. 3595-3608 ◽  
Author(s):  
Anastasia F. Thévenin ◽  
Rachel A. Margraf ◽  
Charles G. Fisher ◽  
Rachael M. Kells-Andrews ◽  
Matthias M. Falk
Keyword(s):  
Protein Kinase ◽  
Gap Junction ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Kinase C ◽  
Zonula Occludens ◽  
Zonula Occludens 1 ◽  
Darby Canine Kidney ◽  
Canine Kidney

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

scholarly journals Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1

2011 ◽  
Vol 22 (9) ◽  
pp. 1516-1528 ◽  
Author(s):  
J. Matthew Rhett ◽  
Jane Jourdan ◽  
Robert G. Gourdie
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cell Coupling ◽  
Protein Protein Interaction ◽  
Dynamic Partitioning ◽  
Zonula Occludens ◽  
Intercellular Channels ◽  
Zonula Occludens 1 ◽  
Interfering Rna

Connexin 43 (Cx43) is a gap junction (GJ) protein widely expressed in mammalian tissues that mediates cell-to-cell coupling. Intercellular channels comprising GJ aggregates form from docking of paired connexons, with one each contributed by apposing cells. Zonula occludens-1 (ZO-1) binds the carboxy terminus of Cx43, and we have previously shown that inhibition of the Cx43/ZO-1 interaction increases GJ size by 48 h. Here we demonstrated that increases in GJ aggregation occur within 2 h (∼Cx43 half-life) following disruption of Cx43/ZO-1. Immunoprecipitation and Duolink protein–protein interaction assays indicated that inhibition targets ZO-1 binding with Cx43 in GJs as well as connexons in an adjacent domain that we term the “perinexus.” Consistent with GJ size increases being matched by decreases in connexons, inhibition of Cx43/ZO-1 reduced the extent of perinexal interaction, increased the proportion of connexons docked in GJs relative to undocked connexons in the plasma membrane, and increased GJ intercellular communication while concomitantly decreasing hemichannel-mediated membrane permeance in contacting, but not noncontacting, cells. ZO-1 small interfering RNA and overexpression experiments verified that loss and gain of ZO-1 function govern the transition of connexons into GJs. It is concluded that ZO-1 regulates the rate of undocked connexon aggregation into GJs, enabling dynamic partitioning of Cx43 channel function between junctional and proximal nonjunctional domains of plasma membrane.

Zonula occludens-1 demonstrates a unique appearance in buccal mucosa over several layers

2021 ◽  
Author(s):  
Keisuke Imafuku ◽  
Mayumi Kamaguchi ◽  
Ken Natsuga ◽  
Hideki Nakamura ◽  
Hiroshi Shimizu ◽  
...  
Keyword(s):  
Buccal Mucosa ◽  
Zonula Occludens ◽  
Zonula Occludens 1

scholarly journals PATH-04. THE BLOOD-BRAIN BARRIER IN DIFFUSE MIDLINE GLIOMA AND ITS IMPLICATIONS FOR DRUG DELIVERY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii164-ii164
Author(s):  
Rianne Haumann ◽  
Fatma El-Khouly ◽  
Marjolein Breur ◽  
Sophie Veldhuijzen van Zanten ◽  
Gertjan Kaspers ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Blood Vessels ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Vascular Density ◽  
Zonula Occludens ◽  
Blood Brain ◽  
Zonula Occludens 1 ◽  
End Stage ◽  
Diffuse Midline Glioma

Abstract INTRODUCTION Chemotherapy has been unsuccessful for pediatric diffuse midline glioma (DMG) most likely due to an intact blood-brain barrier (BBB). However, the BBB has not been characterized in DMG and therefore its implications for drug delivery are unknown. In this study we characterized the BBB in DMG patients and compared this to healthy controls. METHODS End-stage DMG pontine samples (n=5) were obtained from the VUmc diffuse intrinsic pontine glioma (DIPG) autopsy study and age-matched healthy pontine samples (n=22) were obtained from the NIH NeuroBioBank. Tissues were stained for BBB markers claudin-5, zonula occludens-1, laminin, and PDGFRβ. Claudin-5 stains were used to determine vascular density and diameter. RESULTS In DMG, expression of claudin-5 was reduced and dislocated to the abluminal side of endothelial cells. In addition, the expression of zonula occludens-1 was reduced. The basement membrane protein laminin expression was reduced at the glia limitans in both pre-existent vessels and neovascular proliferation. PDGFRβ expression was not observed in DMG but was present in healthy pons. Furthermore, the number of blood vessels in DMG was significantly (P< 0.01) reduced (13.9 ± 11.8/mm2) compared to healthy pons (26.3 ± 14.2/mm2). Markedly, the number of small blood vessels (< 10µm) was significantly lower (P< 0.01) while larger blood vessels (> 10µm) were not significantly different (P= 0.223). The mean vascular diameter was larger for DMG 9.3 ± 9.9µm compared to 7.7 ± 9.0µm for healthy pons (P= 0.016). CONCLUSION Both the BBB and the vasculature are altered at end-stage DMG. The reduced vascular density might have implications for several drug delivery methods such as focused ultrasound and convection enhanced delivery that are being explored for the treatment of DMG. The functional effects of the structurally altered BBB remain unknown and further research is needed to evaluate the BBB integrity at end-stage DMG

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