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

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.

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Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells

Experimental Cell Research ◽

10.1016/j.yexcr.2004.03.021 ◽

2004 ◽

Vol 297 (1) ◽

pp. 247-258 ◽

Cited By ~ 61

Author(s):

Blanca Estela Jaramillo ◽

Arturo Ponce ◽

Jacqueline Moreno ◽

Abigail Betanzos ◽

Miriam Huerta ◽

...

Keyword(s):

Epithelial Cells ◽

Tight Junction ◽

Tight Junction Protein ◽

Junction Protein

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

10.1101/2020.10.04.323436 ◽

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.151.4.825 ◽

2000 ◽

Vol 151 (4) ◽

pp. 825-836 ◽

Cited By ~ 38

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e20-07-0470 ◽

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.

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