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

TLDR

Tight junction strands are composed of claudin‑1, claudin‑2, and occludin. The study introduced FLAG‑tagged claudin‑1, claudin‑2, and occludin cDNAs into TJ‑deficient mouse L fibroblasts to test their ability to form tight junction strands. Mouse L fibroblasts lacking tight junctions were transfected with these cDNAs, and the resulting protein localization and strand formation were examined by immunofluorescence microscopy and freeze‑fracture electron microscopy. Claudin‑1 and claudin‑2 each formed tight junction strands—claudin‑1 produced continuous strands on the protoplasmic face, while claudin‑2 formed discontinuous strands with extracellular grooves—whereas occludin alone formed few strands but was incorporated into claudin‑1 strands when co‑expressed, indicating that claudins are the primary strand components and occludin is an accessory protein.

Abstract

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.