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ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation.

Tornavaca O, Chia M, Dufton N, Almagro LO, Conway DE, Randi AM, Schwartz MA, Matter K, Balda MS - J. Cell Biol. (2015)

Bottom Line: ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex.Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation.ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England, UK.

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ZO-1 regulates the endothelial actomyosin distribution. (A–C) Cells transfected with nontargeting (Control) siRNA or siRNAs directed against ZO-1 were analyzed by immunoblotting for ZO-1 and α-tubulin expression (A), or processed for immunofluorescence microscopy using antibodies against ZO-1 (B) or β-actin, myosin IIA, or double-phosphorylated MLC2 (C). (D) Cells transfected with siRNAs were analyzed by immunoblotting for single- and double-phosphorylated, as well as total, MLC2, and, as a loading control, α-tubulin. (E) Cells that had been transfected with siRNAs as indicated were additionally transfected with GFP or GFP-mZO1, a fusion protein constructed with a mouse ZO-1 cDNA, 24 h before analysis. The cells were then fixed and stained as indicated to monitor loss of stress fibers and increased junctional staining of F-actin and myosin upon ZO-1 reexpression. Bars: (A–C) 40 µm; (E) 20 µm.
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fig1: ZO-1 regulates the endothelial actomyosin distribution. (A–C) Cells transfected with nontargeting (Control) siRNA or siRNAs directed against ZO-1 were analyzed by immunoblotting for ZO-1 and α-tubulin expression (A), or processed for immunofluorescence microscopy using antibodies against ZO-1 (B) or β-actin, myosin IIA, or double-phosphorylated MLC2 (C). (D) Cells transfected with siRNAs were analyzed by immunoblotting for single- and double-phosphorylated, as well as total, MLC2, and, as a loading control, α-tubulin. (E) Cells that had been transfected with siRNAs as indicated were additionally transfected with GFP or GFP-mZO1, a fusion protein constructed with a mouse ZO-1 cDNA, 24 h before analysis. The cells were then fixed and stained as indicated to monitor loss of stress fibers and increased junctional staining of F-actin and myosin upon ZO-1 reexpression. Bars: (A–C) 40 µm; (E) 20 µm.

Mentions: We established a loss-of-function approach to determine the role of ZO-1 in EC using HDMEC. HDMEC were chosen because we found them to form robust and regular junctional complexes. Two distinct siRNAs were identified that effectively down-regulated ZO-1 (Fig. 1, A and B).


ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation.

Tornavaca O, Chia M, Dufton N, Almagro LO, Conway DE, Randi AM, Schwartz MA, Matter K, Balda MS - J. Cell Biol. (2015)

ZO-1 regulates the endothelial actomyosin distribution. (A–C) Cells transfected with nontargeting (Control) siRNA or siRNAs directed against ZO-1 were analyzed by immunoblotting for ZO-1 and α-tubulin expression (A), or processed for immunofluorescence microscopy using antibodies against ZO-1 (B) or β-actin, myosin IIA, or double-phosphorylated MLC2 (C). (D) Cells transfected with siRNAs were analyzed by immunoblotting for single- and double-phosphorylated, as well as total, MLC2, and, as a loading control, α-tubulin. (E) Cells that had been transfected with siRNAs as indicated were additionally transfected with GFP or GFP-mZO1, a fusion protein constructed with a mouse ZO-1 cDNA, 24 h before analysis. The cells were then fixed and stained as indicated to monitor loss of stress fibers and increased junctional staining of F-actin and myosin upon ZO-1 reexpression. Bars: (A–C) 40 µm; (E) 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362456&req=5

fig1: ZO-1 regulates the endothelial actomyosin distribution. (A–C) Cells transfected with nontargeting (Control) siRNA or siRNAs directed against ZO-1 were analyzed by immunoblotting for ZO-1 and α-tubulin expression (A), or processed for immunofluorescence microscopy using antibodies against ZO-1 (B) or β-actin, myosin IIA, or double-phosphorylated MLC2 (C). (D) Cells transfected with siRNAs were analyzed by immunoblotting for single- and double-phosphorylated, as well as total, MLC2, and, as a loading control, α-tubulin. (E) Cells that had been transfected with siRNAs as indicated were additionally transfected with GFP or GFP-mZO1, a fusion protein constructed with a mouse ZO-1 cDNA, 24 h before analysis. The cells were then fixed and stained as indicated to monitor loss of stress fibers and increased junctional staining of F-actin and myosin upon ZO-1 reexpression. Bars: (A–C) 40 µm; (E) 20 µm.
Mentions: We established a loss-of-function approach to determine the role of ZO-1 in EC using HDMEC. HDMEC were chosen because we found them to form robust and regular junctional complexes. Two distinct siRNAs were identified that effectively down-regulated ZO-1 (Fig. 1, A and B).

Bottom Line: ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex.Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation.ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England, UK.

Show MeSH
Related in: MedlinePlus