Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization

The molecular mechanisms of how primordial adherens junctions (AJs) evolve into spatially separated belt-like AJs and tight junctions (TJs) during epithelial polarization are not well understood. Previously, we reported the establishment of ZO-1/ZO-2–deficient cultured epithelial cells (1[ko]/2[kd] cells), which lacked TJs completely. In the present study, we found that the formation of belt-like AJs was significantly delayed in 1(ko)/2(kd) cells during epithelial polarization. The activation of Rac1 upon primordial AJ formation is severely impaired in 1(ko)/2(kd) cells. Our data indicate that ZO-1 plays crucial roles not only in TJ formation, but also in the conversion from “fibroblastic” AJs to belt-like “polarized epithelial” AJs through Rac1 activation. Furthermore, to examine whether ZO-1 itself mediate belt-like AJ and TJ formation, respectively, we performed a mutational analysis of ZO-1. The requirement for ZO-1 differs between belt-like AJ and TJ formation. We propose that ZO-1 is directly involved in the establishment of two distinct junctional domains, belt-like AJs and TJs, during epithelial polarization

Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells

For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier

Backward multiplex coherent anti-Stokes Raman (CARS) spectroscopic imaging with electron-multiplying CCD (EM-CCD) camera

A multiplex CARS imaging system, equipped with an EM-CCD camera, was developed to improve the sensitivity of backward CARS imaging in biological analysis using an inverted microscope. The signal-to-noise ratio was improved by a factor of ca. 3 compared to a conventional CCD mode through the use of EM gain. When imaging epithelial cells in the backward CARS configuration, intracellular organelles such as lipid droplets and nuclei were spectroscopically identified with an exposure time of only 100 ms/pixel.</p

ジョウヒ サイボウ ノ 3サイボウ ケツゴウ Tricellular Junction ニ キョクザイスル シンキ 4カイ マク カンツウ タンパクシツ Tricellulin ノ ハッケン ト ソノ キノウ ニ カンスル ケンキュウ

京都大学0048新制・課程博士博士(医学)甲第12877号医博第3037号新制||医||938(附属図書館)UT51-2007-H150京都大学大学院医学研究科分子医学系専攻(主査)教授 篠原 隆司, 教授 宮地 良樹, 教授 塩田 浩平学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Defining the Roles of β-Catenin and Plakoglobin in LEF/T-Cell Factor-Dependent Transcription Using β-Catenin/Plakoglobin-Null F9 Cells▿

β-Catenin functions as a transcriptional regulator in Wnt signaling. Its function is regulated by a specific destruction system. Plakoglobin is a close homologue of β-catenin in mammalian cells and is regulated in a similar fashion. When β-catenin or plakoglobin is exogenously expressed in cells, endogenous β-catenin is stabilized, which complicates estimation of the transcriptional activities of exogenously expressed proteins. To facilitate the design of experiments aimed at investigating the transcriptional activities of β-catenin and plakoglobin, we utilized F9 cells in which we knocked out endogenous β-catenin and/or plakoglobin by gene deletion and exogenously expressed wild-type and mutant β-catenin and/or plakoglobin. We show that C-terminally deleted β-catenin, but not plakoglobin, has a strong dominant-negative effect on transcription without altering the nuclear accumulation of β-catenin. Moreover, we show that Wnt-3a activation of LEF/T-cell factor (TCF)-dependent transcription depends on β-catenin but not on plakoglobin. Using chimeras of β-catenin and plakoglobin, we demonstrate that plakoglobin has the potential to function in transcriptional regulation but is not responsible for Wnt-3a signaling in F9 cells. Our data show that preferential nuclear accumulation of β-catenin is not necessarily linked to its transcriptional activity. We also clearly demonstrate that plakoglobin is insufficient for LEF/TCF-dependent transcriptional activation by Wnt-3a in F9 cells