Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture

2003 ◽  
Vol 284 (6) ◽  
pp. C1346-C1354 ◽  
Author(s):  
Oscar R. Colegio ◽  
Christina Van Itallie ◽  
Christoph Rahner ◽  
James Melvin Anderson
Keyword(s):  
Transmembrane Proteins ◽  
Extracellular Domain ◽  
Paracellular Permeability ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Ionic Selectivity ◽  
Extracellular Domains ◽  
Charge Selectivity ◽  
Darby Canine Kidney ◽  
Canine Kidney

Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER ∼300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na+ more than Cl− (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319–1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only ∼20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na+ relative to Cl−. In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only ∼60 and ∼40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.

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 Mutations in the Neuraminidase-Like Protein of Bat Influenza H18N11 Virus Enhance Virus Replication in Mammalian Cells, Mice, and Ferrets

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Gongxun Zhong ◽  
Shufang Fan ◽  
Masato Hatta ◽  
Sumiho Nakatsu ◽  
Kevin B. Walters ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Mammalian Cell Lines ◽  
Organ Tropism ◽  
Darby Canine Kidney ◽  
Canine Kidney

ABSTRACT To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets. IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

scholarly journals Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2607-2618 ◽  
Author(s):  
L M Roman ◽  
H Garoff
Keyword(s):  
Semliki Forest Virus ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Directed Transport ◽  
Membrane Spanning ◽  
Darby Canine Kidney ◽  
Canine Kidney

Expression of the Semliki Forest virus p62/E2 protein was studied in the polarized epithelial cell line Madin-Darby canine kidney (MDCK). After infection this transmembrane protein, together with the other spike subunit E1, accumulates at the basolateral surface of MDCK cells (Fuller, S. D., C.-H. von Bonsdorff, and K. Simons, 1985, EMBO (Eur. Mol. Biol. Organ.) J., 4:2475-2485). The cDNAs encoding truncated forms of the protein were used to stably transform MDCK cells to examine the role of subunit oligomerization (E1-E2) and the cytoplasmic domain of p62/E2 in directed transport to the basolateral surface. The biochemical characteristics and polarity of the expressed proteins were studied using cell monolayers grown on nitrocellulose filters. A wild-type form of p62/E2, in the absence of E1, and two forms having either 15 or 3 of the wild-type 31-amino acid carboxyl cytoplasmic domain were all localized to the basolateral surface. These results indicate that the cytoplasmic domain of E2 does not contain the information essential for directed transport to the plasma membrane, and imply that this information resides in either the lumenal and/or membrane-spanning segments of this transmembrane protein.

scholarly journals Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1339-1349 ◽  
Author(s):  
E Camerer ◽  
S Pringle ◽  
AH Skartlien ◽  
M Wiiger ◽  
K Prydz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Tissue Factor ◽  
Factor Vii ◽  
Apical Surface ◽  
Wild Type ◽  
Madin Darby Canine Kidney ◽  
Kidney Epithelial Cells ◽  
Darby Canine Kidney ◽  
Canine Kidney

Tissue factor (TF) is a 48-kD transmembrane glycoprotein that triggers the extrinsic pathway of blood coagulation by interacting with the plasma coagulation factor VII (FVII). TF is also a true receptor in that a cellular signal is generated when activated FVII (FVIIa) binds to TF. For both of these functions, the cellular surface distribution of TF is important, since FVII is primarily available on the apical side of vascular endothelial cells and on the basolateral side of epithelial cells lining the internal and external surfaces. We show that in endothelial cells, TF (both antigen and procoagulant activity) is sorted to the apical surface, whereas in wild-type and stably transfected Madin-Darby canine kidney epithelial cells (MDCK), which form tight junctions and express TF constitutively, TF antigen is on the basolateral surface. No significant clotting activity is detectable on this surface. Truncated TF (cytoplasmic tail residues 246 to 263 deleted) is sorted as wild-type in MDCK cells.

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