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

Abstract

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.

Figure 1.

Retardation of the formation of belt-like AJs and linear actin cables in 1(ko)/2(kd) cells during epithelial polarization. Parental EpH4 cells (A) and 1(ko)/2(kd) cells (B) were cultured overnight in low Ca²⁺ medium overnight, and their polarization was initiated by transferring to normal Ca²⁺ medium. After a 0, 2-, 4-, 8-, or 24-h incubation, cells were fixed and stained with anti-afadin mAb, anti-E cadherin mAb, and phalloidin. Bars, 10 μm.

Figure 2.

Molecular assembly of junctions in 1(ko)/2(kd) cells. (A) Parental EpH4 cells and 1(ko)/2(kd) cells were cocultured and doubly stained with the indicated antibodies 24 h after relpating. Bar, 10 μm. (B) The retardation of the formation of belt-like AJs, TJs, and linear actin cables were canceled by the exogenous expression of GFP-tagged full-length ZO-1. Cells were fixed and double stained with the indicated antibodies 24 h after transfection. Bar, 15 μm.

Figure 3.

Impaired activation of Rac1 in 1(ko)/2(kd) cells during epithelial polarization. (A) Immunoblotting of whole-cell lysates of parental EpH4cells and 1(ko)/2(kd) cells with the indicated antibodies. (B) Parental EpH4 cells and 1(ko)/2(kd) cells were cocultured and doubly stained with the indicated antibodies 24 h after replating. Parental cells are indicated by asterisks. (C) Rac activity assays from parental EpH4 cells and 1(ko)/2(kd) cells after a Ca²⁺ switch for the indicated times. (D) 1(ko)/2(kd) cells were transiently transfected with myc-Rac1-DA or Cdc42-DA. Cells were double stained for myc and anti–nectin -2 antibody (as an AJ marker) or anti–claudin-3 antibody (as a TJ marker) 24 h after transfection. Bars, 10 μm.

Figure 4.

Schematic representation of ZO-1 deletion mutants. (A) Schematic drawings of deletion constructs of ZO-1. Amino acid residues of ZO-1 are shown in parentheses. (B) Total cell lysates derived from HEK293 cells transiently expressing each construct were separated by SDS-PAGE and immunoblotted with anti-GFP pAb. (C) Stable EpH4 lines expressing the deletion constructs of ZO-1 were stained with antibodies against GFP and claudin-3. Note that ZO-1^1-871, ZO-1^516-1746, and ZO-1^804-1746 were incorporated into TJs and did not affect the localization of claudin-3, whereas ZO-1^1-805 was distributed along the lateral plasma membrane and induced ectopic TJs. Bar, 10 μm.

Figure 5.

Rescuing belt-like AJ and/or TJ formation by deletion constructs of ZO-1 in 1(ko)/2(kd) cells. 1(ko)/2(kd) cells were transfected with GFP-tagged ZO-1^1-805 (A), ZO-1^1-871 (B), ZO-1^516-1746 (C), and ZO-1^804-1746 (D). Cells were fixed and stained with the indicated antibodies 24 h after transfection. The formation of belt-like AJs was not observed at cell–cell contacts between ZO-1^1-805 expressing 1(ko)/2(kd) cells (arrowheads). Bar, 10 μm.