The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

Hunter, Andrew W.; Gourdie, Robert G.

doi:10.1080/15419060802014370

Cited by 24 publications

(19 citation statements)

References 42 publications

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“…4a). As has been previously described, ZO-1 localized to the GJ edge and surrounding area (Hunter et al 2005; Hunter and Gourdie 2008; Palatinus et al 2011; Zhu et al 2005). In a result confirming that described by Yoram Rudy and coworkers (Kucera et al 2002), we found a high degree of overlap between the Cx43 and Na v 1.5 signals (Fig.…”

Section: Resultssupporting

confidence: 61%

Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus

et al. 2012

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Gap junctions (GJs) are aggregates of channels that provide for direct cytoplasmic connection between cells. Importantly, this connection is thought responsible for cell-to-cell transfer of the cardiac action potential. The GJ channels of ventricular myocytes are composed of connexin43 (Cx43). Interaction of Cx43 with zonula occludens-1 (ZO-1) is localized not only at the GJ plaque, but also to the region surrounding the GJ, the perinexus. Cx43 in the perinexus is not detectable by immunofluorescence, yet localization of Cx43/ZO-1 interaction to this region indicated the presence of Cx43. Therefore, we hypothesized that Cx43 occurs in the perinexus at a lower concentration per unit membrane than in the GJ itself, making it difficult to visualize. To overcome this, the Duolink protein–protein interaction assay was used to detect Cx43. Duolink labeling of cardiomyocytes localized Cx43 to the perinexus. Quantification demonstrated that signal in the perinexus was lower than in the GJ but significantly higher than in nonjunctional regions. Additionally, Duolink of Triton X-100-extracted cultures suggested that perinexal Cx43 is nonjunctional. Importantly, the voltage gated sodium channel Nav1.5, which is responsible for initiation of the action potential, was found to interact with perinexal Cx43 but not with ZO-1. This work provides a detailed characterization of the structure of the perinexus at the GJ edge and indicates that one of its potential functions in the heart may be in facilitating conduction of action potential.

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Section: Resultssupporting

confidence: 61%

Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus

et al. 2012

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“…10), and by our findings demonstrating the failure of Cx43-EYFP, Cx43⌬363, Cx43⌬257, and Cx43⌬257-EYFP (which do not bind ZO-1) to assemble into GJs. This line of thought is further supported by the studies, which showed that the specific interaction between the PDZ2 domain of ZO-1 and Cx43 was required for GJ assembly but not for trafficking to the cell surface (56,57). Our data also support the conclusions drawn from earlier studies, which showed that the clustering of cell-cell channels to form nascent GJ puncta and their incorporation into the plaques, but not the maintenance of matured GJ plaques, was dependent on an intact actin cytoskeleton (58 -60).…”

Section: Discussionsupporting

confidence: 60%

E-cadherin Differentially Regulates the Assembly of Connexin43 and Connexin32 into Gap Junctions in Human Squamous Carcinoma Cells

Chakraborty

Mitra

Falk

et al. 2010

Journal of Biological Chemistry

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It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.Gap junctions are conglomerations of intercellular channels that permit the direct exchange of ions, second messengers, and other molecules of less than 1500 Da between the cytoplasmic interiors of contiguous cells. The channels are composed of a family of proteins called connexins (Cx) 2 that are designated according to their molecular mass (1). The assembly of Cxs into GJs is a multistep process. The newly synthesized Cxs are cotranslationally inserted into the endoplasmic reticulum, which oligomerize to form hexamers called connexons. The connexons are delivered to the cell surface via Golgi and trans-Golgi network in small vesicles and dock with the connexons in the contiguous cell membranes to form intercellular channels. A GJ plaque is formed when several intercellular channels cluster (2, 3). Because of the short half-life of Cxs (2-5 h), the plaque is in a dynamic state and is thought to model and remodel constantly through recruitment of newly synthesized connexons to the periphery and through endocytosis of moribund plaque components from the center or through the endocytosis of the plaque in its entirety (4 -7). Although much has been learned about the intracellular transport of Cxs, the delivery of connexons to the cell surface, the formation of cell-to-cell channels, the spatio-temporal events and cellular factors required for the initial docking of connexons, the de novo formation of a nascent GJ plaque, its growth, and disassembly are poorly understood (2,8).The proximity between the plasma membranes of adjoining cells is, a priori, necessary for the formation of GJs, and cell-cell adhesi...

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“…How ZO-1 is incorporated into the borders of the gap junctional plaque is not fully understood. However, it has been shown previously that ZO-1 can be localized to gap junctional border zones using a mechanism independent from its direct binding to the C-terminal residues in Cx43 (Hunter and Gourdie, 2008). Given that our current data shows that Vcl and ZO-1 directly interact, we suggest that Vcl might function as a linker anchoring ZO-1 to gap junctions, the actin cytoskeleton and even other common binding partners such as F-actin, a-actinin, and a-catenin.…”

Section: Discussionmentioning

confidence: 99%

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin 43 containing gap junctions in cardiac Myocytes

Zemljic-Harpf

Godoy

Platoshyn

et al. 2014

Journal of Cell Science

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Vinculin (Vcl) links actin filaments to integrin-and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl coimmunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and b1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.

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The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

Cited by 24 publications

References 42 publications

Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus

Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus

E-cadherin Differentially Regulates the Assembly of Connexin43 and Connexin32 into Gap Junctions in Human Squamous Carcinoma Cells

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin 43 containing gap junctions in cardiac Myocytes

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