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© The Rockefeller University Press, 0021-9525/1998//391 $5.00
The Journal of Cell Biology, Volume 143, Number 2, , 1998 391-401


Regular Articles

A Single Gene Product, Claudin-1 or -2, Reconstitutes Tight Junction Strands and Recruits Occludin in Fibroblasts



Mikio Furuse*, Hiroyuki Sasaki{ddagger},§, Kazushi Fujimoto||, and Shoichiro Tsukita*

* Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan; {ddagger} Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan; § Laboratory of Cell Biology, Kan Research Institute Inc., Minami-Kaneda, Suita, Osaka 564, Japan; and || Department of Anatomy, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan

Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1–induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2–induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1–based strands. These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands.

Key Words: tight junction • claudin-1 • claudin-2 • occludin • fibroblast



Abbreviations used in this paper: TJ, tight junction; P face, protoplasmic face; E face, extracellular face; GFP, green fluorescent protein; HA, hemagglutinin; pAb, polyclonal antibody.

Address all correspondence to S. Tsukita, Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Tel.: (81) 75-753-4372. Fax: (81) 75-753-4660. E-mail: htsukita{at}mfour.med.kyoto-u.ac.jp



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