Epiplakin modifies the motility of the HeLa cells and accumulates at the outer surfaces of 3-D cell clusters
Hiromitsu Shimada
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorAkiko Nambu-Niibori
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorMasayo Wilson-Morifuji
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorSouhei Mizuguchi
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorNorie Araki
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorHideaki Sumiyoshi
Department of Matrix Biology, Faculty of Medicine, Oita University, Yufu, Japan
Search for more papers by this authorMitsuru Sato
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan
Search for more papers by this authorYoshihiro Mezaki
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan
Search for more papers by this authorHaruki Senoo
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan
Search for more papers by this authorKazushi Ishikawa
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorYutaka Hatano
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorOsamu Okamoto
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorCorresponding Author
Sakuhei Fujiwara
Department of Dermatology, Oita University, Yufu, Japan
Correspondence: Sakuhei Fujiwara, M.D., Ph.D., Department of Dermatology, Faculty of Medicine, Oita University, Yufu, Japan, Email: [email protected]Search for more papers by this authorHiromitsu Shimada
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorAkiko Nambu-Niibori
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorMasayo Wilson-Morifuji
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorSouhei Mizuguchi
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorNorie Araki
Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorHideaki Sumiyoshi
Department of Matrix Biology, Faculty of Medicine, Oita University, Yufu, Japan
Search for more papers by this authorMitsuru Sato
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan
Search for more papers by this authorYoshihiro Mezaki
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan
Search for more papers by this authorHaruki Senoo
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan
Search for more papers by this authorKazushi Ishikawa
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorYutaka Hatano
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorOsamu Okamoto
Department of Dermatology, Oita University, Yufu, Japan
Search for more papers by this authorCorresponding Author
Sakuhei Fujiwara
Department of Dermatology, Oita University, Yufu, Japan
Correspondence: Sakuhei Fujiwara, M.D., Ph.D., Department of Dermatology, Faculty of Medicine, Oita University, Yufu, Japan, Email: [email protected]Search for more papers by this authorAbstract
Elimination of epiplakin (EPPK) by gene targeting in mice results in acceleration of keratinocyte migration during wound healing, suggesting that epithelial cellular EPPK may be important for the regulation of cellular motility. To study the function of EPPK, we developed EPPK knock-down (KD) and EPPK-overexpressing HeLa cells and analyzed cellular phenotypes and motility by fluorescence/differential interference contrast time-lapse microscopy and immunolocalization of actin and vimentin. Cellular motility of EPPK-KD cells was significantly elevated, but that of EPPK-overexpressing cells was obviously depressed. Many spike-like projections were observed on EPPK-KD cells, with fewer such structures on overexpressing cells. By contrast, in EPPK-KD cells, expression of E-cadherin was unchanged but vimentin fibers were thinner and sparser than in controls, and they were more concentrated at the peri-nucleus, as observed in migrating keratinocytes at wound edges in EPPK−/− mice. In Matrigel 3-D cultures, EPPK co-localized on the outer surface of cell clusters with zonula occludens-1 (ZO-1), a marker of tight junctions. Our results suggest that EPPK is associated with the machinery for cellular motility and contributes to tissue architecture via the rearrangement of intermediate filaments.
Supporting Information
| Filename | Description |
|---|---|
| jde12076-sup-0001-FigS1.tifimage/tif, 1.1 MB | Figure S1. Double immunostaining with anti-epiplakin (EPPK) antibodies (green) and anti-keratin 8/18 (a, red) or anti-vimentin (b, red) antibodies.(a; stained also with 4′,6′-diamidino-2-phenylindole dihydrochloride) Endogeneous EPPK co-localized with Intermediate Filaments network and it co-localized more closely with the keratin network than with vimentin in HeLa cells. Bar, 10 μm. |
| jde12076-sup-0002-FigS2.tifimage/tif, 993.4 KB | Figure S2. We stained HeLa cell clusters in 3-D culture in Matrigel using anti-BP230 antibodies (sc13777). One of the hemidesmosomal proteins, BP230, not only co-localized with epiplakin (EPPK) partly on the outermost surface of the cell clusters, but also located inside cell clusters. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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