scholarly journals Exogenous Expression of the Amino-Terminal Half of the Tight Junction Protein Zo-3 Perturbs Junctional Complex Assembly

2000 ◽  
Vol 151 (4) ◽  
pp. 825-836 ◽  
Author(s):  
Erika S. Wittchen ◽  
Julie Haskins ◽  
Bruce R. Stevenson
Keyword(s):  
Tight Junction ◽  
Actin Cytoskeleton ◽  
Mdck Cells ◽  
Dominant Negative ◽  
Tight Junction Protein ◽  
Junctional Complex ◽  
Amino Terminal ◽  
Junction Protein ◽  
Exogenous Expression ◽  
E Cadherin

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and β-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100–soluble, signaling-active β-catenin was increased in NZO-3–expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or β-catenin.

scholarly journals NZO-3 Expression Causes Global Changes to Actin Cytoskeleton in Madin-Darby Canine Kidney Cells: Linking a Tight Junction Protein to Rho GTPases

2003 ◽  
Vol 14 (5) ◽  
pp. 1757-1768 ◽  
Author(s):  
Erika S. Wittchen ◽  
Julie Haskins ◽  
Bruce R. Stevenson
Keyword(s):  
Tight Junction ◽  
Actin Cytoskeleton ◽  
Tight Junction Protein ◽  
Junctional Complex ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
P120 Catenin ◽  
Junction Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

We previously demonstrated that exogenous expression of a truncated form of the tight junction protein ZO-3 affected junctional complex assembly and function. Current results indicate that this ZO-3 construct influences actin cytoskeleton dynamics more globally. We show that expression of the amino-terminal half of ZO-3 (NZO-3) in Madin-Darby canine kidney cells results in a decreased number of stress fibers and focal adhesions and causes an increased rate of cell migration in a wound healing assay. We also demonstrate that RhoA activity is reduced in NZO-3–expressing cells. We determined that ZO-3 interacts with p120 catenin and AF-6, proteins localized to the junctional complex and implicated in signaling pathways important for cytoskeleton regulation and cell motility. We also provide evidence that NZO-3 interacts directly with the C terminus of ZO-3, and we propose a model where altered interactions between ZO-3 and p120 catenin in NZO-3–expressing cells affect RhoA GTPase activity. This study reveals a potential link between ZO-3 and RhoA-related signaling events.

scholarly journals Correlation of occludin protein mobility with paracellular leak pathway permeability in renal epithelia

2018 ◽  
Author(s):  
Josephine Axis ◽  
Alexander L. Kolb ◽  
Robert L. Bacallao ◽  
Kurt Amsler
Keyword(s):  
Tight Junction ◽  
Ischemia Reperfusion Injury ◽  
Mdck Cells ◽  
Ischemia Reperfusion ◽  
Paracellular Permeability ◽  
Tight Junction Protein ◽  
Protein Mobility ◽  
Junction Protein ◽  
Leak Pathway

ABSTRACTStudies have demonstrated regulation of the epithelial paracellular permeability barrier, the tight junction, by a variety of stimuli. Recent studies have reported a correlation between changes in paracellular permeability, particularly paracellular permeability to large solutes (leak pathway), and mobility of the tight junction protein, occludin, in the plane of the plasma membrane. This had led to the hypothesis that changes in occludin protein mobility are causative for changes in paracellular permeability. Using a renal epithelial cell model system, MDCK, we examined the effect of various manipulations on both leak pathway permeability, monitored as the paracellular movement of a fluorescent molecule (calcein), and occludin protein mobility, monitored through fluorescence recovery after photobleaching. Our results indicate that knockdown of the associated tight junction protein, ZO-1, increases baseline leak pathway permeability, whereas, knockdown of the related tight junction protein, ZO-2, does not alter baseline leak pathway permeability. Knockdown of either ZO-1 or ZO-2 decreases the rate of movement of occludin protein but only knockdown of ZO-2 protein alters the percent of occludin protein that is mobile. Further, treatment with hydrogen peroxide increases leak pathway permeability in wild type MDCK cells and in ZO-2 knockdown MDCK cells but not in ZO-1 knockdown MDCK cells. This treatment decreases the rate of occludin movement in all three cell lines but only alters the mobile fraction of occludin protein in ZO-1 knockdown MDCK cells. Finally, we examined the effect of renal ischemia/reperfusion injury on occludin protein mobility in vivo.Ischemia/reperfusion injury both increased the rate of occludin mobility and increased the fraction of occludin protein that is mobile. These results indicate that, at least in our cell culture and in vivo model systems, there is no consistent correlation between paracellular leak pathway permeability and occludin protein mobility.

Molecular Characterization of the Tight Junction Protein ZO-1 in MDCK Cells

1999 ◽  
Vol 248 (1) ◽  
pp. 97-109 ◽  
Author(s):  
L. González-Mariscal ◽  
S. Islas ◽  
R.G. Contreras ◽  
M.R. Garcı́a-Villegas ◽  
A. Betanzos ◽  
...  
Keyword(s):  
Tight Junction ◽  
Molecular Characterization ◽  
Mdck Cells ◽  
Tight Junction Protein ◽  
Junction Protein

scholarly journals Force-dependent remodeling of a tight junction protein ZO-1 is regulated by phase separation

2020 ◽  
Author(s):  
Noriyuki Kinoshita ◽  
Takamasa S. Yamamoto ◽  
Naoko Yasue ◽  
Toshihiko Fujimori ◽  
Naoto Ueno
Keyword(s):  
Phase Separation ◽  
Tight Junction ◽  
Inner Cell Mass ◽  
Mdck Cells ◽  
Cell Mass ◽  
Tight Junction Protein ◽  
Cell Junction ◽  
Physiological Importance ◽  
Junction Protein ◽  
Tensile Forces

SummaryAlthough the physiological importance of biomolecular condensates is widely recognized, how it is controlled in time and space during development is largely unknown. Here we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows, and internal pressure on TE cells increases. In contrast, the dissolution is less evident in TE cells attached to the inner cell mass, as they receive weaker tensile forces. Furthermore, analyses using MDCK cells have shown that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via the interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to establishing robust cell-cell adhesion during early development.

Tight junction protein, claudin 4 but not ZO-1 expression is reduced along with E-cadherin in diffuse type gastric adenocarcinoma

2003 ◽  
Vol 124 (4) ◽  
pp. A555
Author(s):  
Sang Kil Lee ◽  
Seung Woo Park ◽  
Si Young Song ◽  
Jae Bock Chung ◽  
Jin Kyung Kang
Keyword(s):  
Tight Junction ◽  
Gastric Adenocarcinoma ◽  
Tight Junction Protein ◽  
Diffuse Type ◽  
Junction Protein ◽  
E Cadherin

Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly

2000 ◽  
Vol 113 (6) ◽  
pp. 985-996 ◽  
Author(s):  
M.L. Troxell ◽  
S. Gopalakrishnan ◽  
J. McCormack ◽  
B.A. Poteat ◽  
J. Pennington ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tight Junction ◽  
Mdck Cells ◽  
Freeze Fracture ◽  
Junctional Complex ◽  
Negative Effects ◽  
Tight Junction Formation ◽  
Freeze Fracture Electron Microscopy ◽  
E Cadherin ◽  
Cell Cell

Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.

scholarly journals The Tight Junction Protein ZO-1 Establishes a Link between the Transmembrane Protein Occludin and the Actin Cytoskeleton

1998 ◽  
Vol 273 (45) ◽  
pp. 29745-29753 ◽  
Author(s):  
Alan S. Fanning ◽  
Brian J. Jameson ◽  
Lynne A. Jesaitis ◽  
James Melvin Anderson
Keyword(s):  
Tight Junction ◽  
Actin Cytoskeleton ◽  
Transmembrane Protein ◽  
Tight Junction Protein ◽  
Junction Protein

scholarly journals Tight junction protein ZO-2 modulates the nuclear accumulation of transcription factor TEAD

2021 ◽  
pp. mbc.E20-07-0470
Author(s):  
Helios Gallego-Gutiérrez ◽  
Laura González-González ◽  
Leticia Ramírez-Martínez ◽  
Esther López-Bayghen ◽  
Lorenza González-Mariscal
Keyword(s):  
Transcription Factor ◽  
Tight Junction ◽  
Nuclear Export ◽  
Mdck Cells ◽  
Tight Junction Protein ◽  
Nuclear Accumulation ◽  
Intracellular Traffic ◽  
Nuclear Localization Signals ◽  
Proximity Ligation ◽  
Junction Protein

Tight junction protein Zonula occludens 2 (ZO-2) presence at the nucleus inhibits the transcription of genes regulated by TEAD transcription factor. Here, we analyzed if the movement of ZO-2 into the nucleus modulates the nuclear concentration of TEAD. In sparse cultures of ZO-2 knock down (KD) MDCK cells, nuclear TEAD diminished, as in parental cells transfected with a ZO-2 construct without nuclear localization signals, indicating that ZO-2 facilitates the entry of TEAD into the nucleus. Inhibition of nPKCδ in parental cells triggers the interaction between ZO-2 and TEAD at the cytoplasm and facilitates TEAD/ZO-2 complex nuclear importation. Using proximity ligation, immunoprecipitation and pull-down assays TEAD/ZO-2 interaction was confirmed. Nuclear TEAD is phosphorylated and its exit in parental cells is enhanced by activation of a ZO-2 nuclear exportation signal by nPKCε, while the nuclear accumulation of ZO-2 triggered by the mutation of ZO-2 nuclear export signals, induces the no change in TEAD nuclear concentration. In summary, our results indicate that the movements of ZO-2 in and out of the nucleus modulate the intracellular traffic of TEAD through a process regulated by nPKC δ and ε, and provide a novel role of ZO-2 as a nuclear translocator of TEAD.

scholarly journals Differential regulation of junctional complex assembly in renal epithelial cell lines

2003 ◽  
Vol 285 (1) ◽  
pp. C102-C111 ◽  
Author(s):  
Shobha Gopalakrishnan ◽  
Mark A. Hallett ◽  
Simon J. Atkinson ◽  
James. A. Marrs
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Rho Gtpase ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Stress Fiber ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Complex Assembly ◽  
E Cadherin

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and β-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.

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