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CPEB-mediated ZO-1 mRNA localization is required for epithelial tight-junction assembly and cell polarity.

Nagaoka K, Udagawa T, Richter JD - Nat Commun (2012)

Bottom Line: CPEB and zonal occludens-1 mRNA are co-localized apically and zonal occludens-1 3' untranslated region-binding sites for CPEB are necessary for RNA localization.In a three-dimensional culture system that models lumen-containing mammary ducts, depletion of CPEB or zonal occludens-1 impairs central cavity formation, indicating a loss of cell polarity.Cavity formation in zonal occludens-1-depleted cells is rescued when they are transduced with zonal occludens-1 mRNA containing, but not lacking, CPEB-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

ABSTRACT
CPEB is a translational regulatory sequence-specific RNA-binding protein that controls germ cell development. Here we show that CPEB heterozygous female mice are fertile but contain disorganized mammary epithelial cells, in which zonal occludens-1 and claudin-3, apical tight-junction proteins, are mislocalized. CPEB depletion from mammary epithelial cells disrupts zonal occludens-1 apical localization and tight-junction distribution; conversely, ectopic expression of CPEB enhances zonal occludens-1 localization. CPEB and zonal occludens-1 mRNA are co-localized apically and zonal occludens-1 3' untranslated region-binding sites for CPEB are necessary for RNA localization. In a three-dimensional culture system that models lumen-containing mammary ducts, depletion of CPEB or zonal occludens-1 impairs central cavity formation, indicating a loss of cell polarity. Cavity formation in zonal occludens-1-depleted cells is rescued when they are transduced with zonal occludens-1 mRNA containing, but not lacking, CPEB-binding sites. Our data demonstrate that CPEB-mediated zonal occludens-1 mRNA localization is essential for tight-junction assembly and mammary epithelial cell polarity.

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ZO-1 localization to apical tight junctions and cavity formation require CPEB(a) EpH4 cells were grown in suspension in matrigel for 3–12 days. Cavity formation was monitored by immunostaining for ZO-1 and E-cadherin and examined by confocal microscopy. Scale bar = 10µm. (b) Analysis of cavity formation in EpH4 cells cultured in matrigel for 12 days. Scale bar is 10 µm. The histogram shows the percent of colonies with cavities following the indicated treatments. The two asterisks refer to statistical significance (p<0.01, Student’s t test, 3 replicates). (c) FLAG-CPEB as well as ZO-1 expression in cells that form cavities. Mock refers to cells expressing FLAG only (a nonsense protein that terminated at a stop codon in vector sequences). Scale bar is 10 µm. The histogram shows quantification of FLAG in the apical portion of cells. The asterisks refers to statistical significance (p<0.01, Student’s t test, 3 replicates). Error bars are standard error of the mean.
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Figure 4: ZO-1 localization to apical tight junctions and cavity formation require CPEB(a) EpH4 cells were grown in suspension in matrigel for 3–12 days. Cavity formation was monitored by immunostaining for ZO-1 and E-cadherin and examined by confocal microscopy. Scale bar = 10µm. (b) Analysis of cavity formation in EpH4 cells cultured in matrigel for 12 days. Scale bar is 10 µm. The histogram shows the percent of colonies with cavities following the indicated treatments. The two asterisks refer to statistical significance (p<0.01, Student’s t test, 3 replicates). (c) FLAG-CPEB as well as ZO-1 expression in cells that form cavities. Mock refers to cells expressing FLAG only (a nonsense protein that terminated at a stop codon in vector sequences). Scale bar is 10 µm. The histogram shows quantification of FLAG in the apical portion of cells. The asterisks refers to statistical significance (p<0.01, Student’s t test, 3 replicates). Error bars are standard error of the mean.

Mentions: EpH4 cells grown in an anchorage-independent manner are polarized and contain central cavities; consequently, they have been used as in vitro models to examine mammary duct formation28. As expected, ZO-1 was detected in the apical region (Fig 4a, b). Cavity formation was strongly reduced when CPEB was depleted, but was rescued when CPEB was ectopically expressed (Fig. 4b). In CPEB-depleted colonies that did not form cavities, ZO-1 was randomly distributed even though steady state levels were uanffected.


CPEB-mediated ZO-1 mRNA localization is required for epithelial tight-junction assembly and cell polarity.

Nagaoka K, Udagawa T, Richter JD - Nat Commun (2012)

ZO-1 localization to apical tight junctions and cavity formation require CPEB(a) EpH4 cells were grown in suspension in matrigel for 3–12 days. Cavity formation was monitored by immunostaining for ZO-1 and E-cadherin and examined by confocal microscopy. Scale bar = 10µm. (b) Analysis of cavity formation in EpH4 cells cultured in matrigel for 12 days. Scale bar is 10 µm. The histogram shows the percent of colonies with cavities following the indicated treatments. The two asterisks refer to statistical significance (p<0.01, Student’s t test, 3 replicates). (c) FLAG-CPEB as well as ZO-1 expression in cells that form cavities. Mock refers to cells expressing FLAG only (a nonsense protein that terminated at a stop codon in vector sequences). Scale bar is 10 µm. The histogram shows quantification of FLAG in the apical portion of cells. The asterisks refers to statistical significance (p<0.01, Student’s t test, 3 replicates). Error bars are standard error of the mean.
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Related In: Results  -  Collection

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Figure 4: ZO-1 localization to apical tight junctions and cavity formation require CPEB(a) EpH4 cells were grown in suspension in matrigel for 3–12 days. Cavity formation was monitored by immunostaining for ZO-1 and E-cadherin and examined by confocal microscopy. Scale bar = 10µm. (b) Analysis of cavity formation in EpH4 cells cultured in matrigel for 12 days. Scale bar is 10 µm. The histogram shows the percent of colonies with cavities following the indicated treatments. The two asterisks refer to statistical significance (p<0.01, Student’s t test, 3 replicates). (c) FLAG-CPEB as well as ZO-1 expression in cells that form cavities. Mock refers to cells expressing FLAG only (a nonsense protein that terminated at a stop codon in vector sequences). Scale bar is 10 µm. The histogram shows quantification of FLAG in the apical portion of cells. The asterisks refers to statistical significance (p<0.01, Student’s t test, 3 replicates). Error bars are standard error of the mean.
Mentions: EpH4 cells grown in an anchorage-independent manner are polarized and contain central cavities; consequently, they have been used as in vitro models to examine mammary duct formation28. As expected, ZO-1 was detected in the apical region (Fig 4a, b). Cavity formation was strongly reduced when CPEB was depleted, but was rescued when CPEB was ectopically expressed (Fig. 4b). In CPEB-depleted colonies that did not form cavities, ZO-1 was randomly distributed even though steady state levels were uanffected.

Bottom Line: CPEB and zonal occludens-1 mRNA are co-localized apically and zonal occludens-1 3' untranslated region-binding sites for CPEB are necessary for RNA localization.In a three-dimensional culture system that models lumen-containing mammary ducts, depletion of CPEB or zonal occludens-1 impairs central cavity formation, indicating a loss of cell polarity.Cavity formation in zonal occludens-1-depleted cells is rescued when they are transduced with zonal occludens-1 mRNA containing, but not lacking, CPEB-binding sites.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

ABSTRACT
CPEB is a translational regulatory sequence-specific RNA-binding protein that controls germ cell development. Here we show that CPEB heterozygous female mice are fertile but contain disorganized mammary epithelial cells, in which zonal occludens-1 and claudin-3, apical tight-junction proteins, are mislocalized. CPEB depletion from mammary epithelial cells disrupts zonal occludens-1 apical localization and tight-junction distribution; conversely, ectopic expression of CPEB enhances zonal occludens-1 localization. CPEB and zonal occludens-1 mRNA are co-localized apically and zonal occludens-1 3' untranslated region-binding sites for CPEB are necessary for RNA localization. In a three-dimensional culture system that models lumen-containing mammary ducts, depletion of CPEB or zonal occludens-1 impairs central cavity formation, indicating a loss of cell polarity. Cavity formation in zonal occludens-1-depleted cells is rescued when they are transduced with zonal occludens-1 mRNA containing, but not lacking, CPEB-binding sites. Our data demonstrate that CPEB-mediated zonal occludens-1 mRNA localization is essential for tight-junction assembly and mammary epithelial cell polarity.

Show MeSH
Related in: MedlinePlus