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Volume 35 (2010)
- Issue 2 Pages 73-
- Issue 1 Pages 1-
Volume 35, Issue 2
Displaying 1-4 of 4 articles from this issue
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Nobuaki Shiraki, Seiko Harada, Soichiro Ogaki, Kazuhiko Kume, Shoen Ku ...2010 Volume 35 Issue 2 Pages 73-80
Published: 2010
Released on J-STAGE: July 01, 2010
Advance online publication: June 04, 2010JOURNAL FREE ACCESS FULL-TEXT HTML
Supplementary materialAlthough there are several markers available for the identification of endoderm derivatives such as the lung, pancreas, liver, and intestine, there are still very few surface markers available for the prospective isolation of the definitive endoderm. Among these, CXCR4 has been used in combination with E-cadherin as a cell surface marker to identify the definitive endoderm. However, CXCR4 expression decreases in late gut epithelium. Here we report a gene, Decay Accelerating Factor (DAF1/CD55), as a candidate surface marker for the identification of the early and late definitive endoderm. Daf1 is expressed in the definitive endoderm and mesoderm in early embryo at E8.5. Flow cytometry analysis of ES cells-derived differentiated cells revealed that DAF1-expressing cells also expressed CXCR4. Moreover, DAF1 expression is maintained until differentiation day 12 in ES cell-derived definitive endoderm cells. Analysis of the Pdx1/GFP-positive cells in E9.5 embryos and ES cell-derived cells with anti-DAF1 revealed that most Pdx1-GFP cells expressed DAF1. These results suggest that DAF1, when used in combination with E-cadherin, is useful for prospective identification of the definitive endoderm cells.
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Chisa Ozaki, Masato Yoshioka, Sachiko Tominaga, Yoshinori Osaka, Shuic ...2010 Volume 35 Issue 2 Pages 81-94
Published: 2010
Released on J-STAGE: October 22, 2010
Advance online publication: September 16, 2010JOURNAL FREE ACCESS FULL-TEXT HTML
Supplementary materialThe role of p120-catenin in the function of classical cadherins is still enigmatic despite various studies. To elucidate its role, we examined the effect of p120-catenin on the N-cadherin-mediated localization of junctional proteins in epithelial cells in this study. Cadherin-deficient MIA PaCa-2 epithelial cells did not show linear localization of tight junction proteins ZO-1 and occludin. When N-cadherin was expressed in these cells, however, the resultant transfectant cells revealed strong cell adhesion activity and linear localization of ZO-1, occludin, and N-cadherin in the lateral membrane. When the p120-catenin-binding site of N-cadherin was disrupted, the linear localization of ZO-1 and occludin disappeared, and the mutant N-cadherin became localized more diffusely in the transfectant, although the cell adhesion activity did not change much. Knockdown of p120-catenin also resulted in the very weak localization of ZO-1 and occludin. A similar effect of p120-catenin on the localization of junctional proteins was obtained under more dynamic conditions in a wound healing assay. Moreover, p120-catenin was essential for the regulation of centrosome orientation in this healing assay. Taken together, the present data indicate that p120-catenin is essential for N-cadherin-mediated formation of proper junctional structures and thereby the establishment of the cell polarity. Similar results were obtained when E-cadherin mutants comparable to those of N-cadherin were used, suggesting that p120-catenin plays the same role in the function of other classical cadherins.
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Naoko Mauchi, Yoshiaki Ohtake, Kenji Irie2010 Volume 35 Issue 2 Pages 95-105
Published: 2010
Released on J-STAGE: November 17, 2010
Advance online publication: October 13, 2010JOURNAL FREE ACCESS FULL-TEXT HTMLKhd1p (KH-domain protein 1) is a yeast RNA-binding protein highly homologous to mammalian hnRNP K. Khd1p associates with hundreds of potential mRNA targets including a bud-localized ASH1 mRNA and mRNAs encoding membrane-associated proteins such as Mid2p and Mtl1p. While Khd1p negatively regulates gene expression of Ash1p by translational repression, Khd1p positively regulates gene expression of Mtl1p by mRNA stabilization. To investigate how Khd1p regulates the stability of MTL1 mRNA, we searched for cis-acting elements and trans-acting factors controlling MTL1 mRNA stability. Regional analysis revealed that partial deletion of the coding sequences of MTL1 mRNA restored the decreased MTL1 mRNA and protein levels in khd1Δ mutants. This region, encompassing nucleotides 532 to 1032 of the Mtl1p coding sequence, contains CNN repeats that direct Khd1p-binding. Insertion of this sequence into other mRNAs conferred mRNA instability in khd1Δ mutants. We further searched for factors involved in the destabilization of MTL1 mRNA. Mutations in CCR4 and CAF1/POP2, encoding major cytoplasmic deadenylases, or of SKI genes, which code for components of a complex involved in 3' to 5' degradation, did not restore the decreased MTL1 mRNA levels caused by khd1Δ mutation. However, mutations in DCP1 and DCP2, encoding a decapping enzyme complex, and XRN1, encoding a 5'-3' exonuclease, restored the decreased MTL1 mRNA levels. Furthermore, Khd1p colocalized with Dcp1p in processing bodies, cytoplasmic sites for mRNA degradation. Our results suggest that MTL1 mRNA bears a cis-acting element involved in destabilization by the decapping enzyme and the 5'-3' exonuclease, and Khd1p stabilizes MTL1 mRNA through binding to this element.
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Yo-hei Yamamoto, Taiji Kimura, Shuku Momohara, Masato Takeuchi, Tokio ...2010 Volume 35 Issue 2 Pages 107-116
Published: 2010
Released on J-STAGE: December 23, 2010
Advance online publication: December 08, 2010JOURNAL FREE ACCESS FULL-TEXT HTMLCytosolic Hsc70/Hsp70 are known to contribute to the endoplasmic reticulum (ER)-associated degradation of membrane proteins. However, at least in mammalian cells, its partner ER-localized J-protein for this cellular event has not been identified. Here we propose that this missing protein is DNAJB12. Protease protection assay and immunofluorescence study revealed that DNAJB12 is an ER-localized single membrane-spanning protein carrying a J-domain facing the cytosol. Using co-immunoprecipitation assay, we found that DNAJB12 is able to bind Hsc70 and thus can recruit Hsc70 to the ER membrane. Remarkably, cellular overexpression of DNAJB12 accelerated the degradation of misfolded membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR), but not a misfolded luminal protein. The DNAJB12-dependent degradation of CFTR was compromised by a proteasome inhibitor, lactacystin, suggesting that this process requires the ubiquitin-proteasome system. Conversely, knockdown of DNAJB12 expression attenuated the degradation of CFTR. Thus, DNAJB12 is a novel mammalian ER-localized J-protein that plays a vital role in the quality control of membrane proteins.
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