Adherens junctions influence tight junction formation via changes in membrane lipid composition

Abstract

Tight junctions (TJs) are essential cell adhesion structures that act as a barrier to separate the internal milieu from the external environment in multicellular organisms. Although their major constituents have been identified, it is unknown how the formation of TJs is regulated. TJ formation depends on the preceding formation of adherens junctions (AJs) in epithelial cells; however, the underlying mechanism remains to be elucidated. In this study, loss of AJs in α-catenin-knockout (KO) EpH4 epithelial cells altered the lipid composition of the plasma membrane (PM) and led to endocytosis of claudins, a major component of TJs. Sphingomyelin with long-chain fatty acids and cholesterol were enriched in the TJ-containing PM fraction. Depletion of cholesterol abolished the formation of TJs. Conversely, addition of cholesterol restored TJ formation in α-catenin-KO cells. Collectively, we propose that AJs mediate the formation of TJs by increasing the level of cholesterol in the PM.

Figure 1.

α-Catenin–KO cells internalize claudins. (A) Phase-contrast images of WT and α-catenin–KO EpH4 cells. (B) Immunoblotting of whole-cell lysates of WT and α-catenin–KO EpH4 cells with the indicated antibodies. (C) WT and α-catenin–KO EpH4 cells were fixed and costained with an anti–claudin-3 pAb and an anti–E-cadherin mAb (left) or with an anti–JAM-A pAb and an antioccludin mAb (right). (D) α-Catenin–KO EpH4 cells stably expressing GFP-tagged mouse α-catenin were fixed and costained with an anti–claudin-3 pAb and an anti–E-cadherin mAb. (E) Immunoblotting of whole-cell lysates of WT EpH4 cells, α-catenin–KO EpH4 cells, and α-catenin–KO EpH4 cells stably expressing GFP-tagged α-catenin (rescue) with the indicated antibodies. Molecular masses are given in kilodaltons. (F) α-Catenin–KO EpH4 cells were fixed and costained with an anti–claudin-3 pAb (green) and an anti-EEA1 mAb (red, top), an anti-LAMP1 mAb (red, middle), or an anti-GM130 mAb (red, bottom). Arrowheads indicate colocalization. (G) α-Catenin–KO EpH4 cells were treated with DMSO (control, top), 10 µg/ml chlorpromazine (middle) for 1 h, or 100 µM dynasore (bottom) for 2 h, fixed, and stained with an anti–claudin-3 pAb. Bars: (A, C, D, and F) 20 µm; (G) 25 µm.

Figure 2.

The level of cholesterol is reduced in the PM of α-catenin–KO cells. (A) Positive ion mass spectra of SM species in WT and α-catenin–KO EpH4 cells. The SM molecular species corresponding with each peak are indicated. The x and y axes show the total carbon chain length and the number of carbon–carbon double bonds of individual lipid molecular species, respectively. The results are representative of three independent experiments. (B) Quantification of the indicated SM species in WT EpH4 cells and α-catenin–KO EpH4 cells. Error bars show SD calculated based on three independent experiments (Student’s t test, *, P < 0.05). (C) WT and α-catenin–KO EpH4 cells were fixed with 4% paraformaldehyde and stained with 50 µg/ml filipin prepared in PBS to visualize the subcellular localization of cholesterol. (D) WT EpH4 cells expressing GFP–claudin-3 were fixed with 4% paraformaldehyde and stained with 50 µg/ml filipin prepared in PBS. (E) Confluent WT EpH4 cells expressing GFP–claudin-3 were cultured in low-Ca²⁺ medium containing 5 µM Ca²⁺ overnight to disrupt AJs completely (left) and then in normal Ca²⁺ medium containing ECCD-1 (1:500 dilution) for 1 h (middle). Thereafter, ECCD-1 was washed out, and cells were cultured in normal Ca²⁺ medium for 1 h (right). After fixation with 4% paraformaldehyde, cells were stained with 50 µg/ml filipin prepared in PBS. Bars, 20 µm.

Figure 3.

Cholesterol is enriched in the TJ-containing PM fraction. (A) C1L cells were fixed and stained with an anti–claudin-1 pAb. (B) C1L cells were fixed and stained with an anti–claudin-1 pAb (green) and phalloidin (red). Bars, 10 µm. (C) Immunoblot analysis of the PM and IM fractions of C1L cells. Each membrane fraction (5 µg) was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies against the indicated marker proteins (left). Coomassie brilliant blue (CBB) staining is shown on the right. (D) Positive ion mass spectra of SM species in the PM fractions of L and C1L cells. The SM molecular species corresponding with each peak are indicated. The x and y axes show the total carbon chain length and the number of carbon–carbon double bonds of individual lipid molecular species, respectively. (E) Quantification of the indicated SM species in the PM fractions of L cells and C1L cells. (F) Quantification of the cholesterol-to-phospholipid ratio in the PM fractions of L and C1L cells. (G) Immunoblot analysis of the DRM and non-DRM fractions of WT and α-catenin–KO EpH4 cells using pAbs against the DRM marker proteins claudin-3 and caveolin-1. Results in C, D, and G are representative of three independent experiments. (H) Quantification of the ratio of the claudin-3 level in the DRM fraction to that in the non-DRM fraction in WT and α-catenin–KO EpH4 cells. Error bars show SD calculated based on three independent experiments (Student’s t test, *, P < 0.05).

Figure 4.

Depletion of cholesterol specifically impairs the formation of TJs. (A) WT EpH4 cells were cultured in transwell chambers and treated with PBS (control) or 75 mM MβCD for the indicated duration, and then cells underwent TER analysis (means ± SD; n = 4). (B) WT EpH4 cells were treated with PBS (control), 25 mM MβCD, 50 mM MβCD, or 75 mM MβCD for 30 min, fixed, and costained with an anti–claudin-3 pAb and an anti–E-cadherin mAb. (C) WT EpH4 cells were treated with PBS (control) or 75 mM MβCD for 30 min, fixed, and costained with an anti–claudin-3 pAb and an anti–desmoglein-2 mAb. (D) WT EpH4 cells were treated with PBS (control) or 50 mM MβCD for 30 min, fixed, and costained with an anti–E-cadherin mAb and an antioccludin pAb. Bars, 20 µm.

Figure 5.

Addition of cholesterol to the PM induces TJ strand formation in α-catenin–KO cells. (A) Time-lapse imaging of α-catenin–KO EpH4 cells expressing GFP–claudin-3. At time 0, 75 mM cholesterol-saturated MβCD was added to the medium to restore the level of cholesterol in the PM. (B) α-Catenin–KO EpH4 cells were treated with PBS (control) or 75 mM cholesterol-saturated MβCD, fixed, and costained with an anti–claudin-3 pAb (green) and an anti–ZO-1 mAb (red). (C) Quantification of the signal intensity of claudin-3 at cell–cell contact areas in α-catenin–KO EpH4 cells before and after loading of cholesterol in the PM. (D) Quantification of the colocalization of claudin-3 and ZO-1 in α-catenin–KO EpH4 cells before and after loading of cholesterol in the PM. The degree of colocalization between claudin-3 and ZO-1 was calculated using ImageJ FIJI software. The value of Pearson’s coefficient of two signals were quantitated. Error bars show SD calculated based on four independent experiments (Student’s t test, *, P < 0.05). (E) Freeze-fracture EM images of TJ strands in α-catenin–KO EpH4 cells treated with PBS (control, top) or 75 mM cholesterol-saturated MβCD (bottom) for 30 min. (F) α-Catenin–KO EpH4 cells were treated 75 mM cholesterol-saturated MβCD, fixed, and stained with an anti–claudin-3 pAb (green) together with an anti–E-cadherin mAb (red, top) or an antivinculin mAb (red, bottom). (G) α-Catenin–KO EpH4 cells expressing GFP–claudin-3 were treated with 75 mM cholesterol-saturated MβCD, fixed with 4% paraformaldehyde, and stained with 50 µg/ml filipin prepared in PBS. (H) α-Catenin–KO EpH4 cells expressing GFP–claudin-3 were treated with DMSO (control, top) or 100 µM dynasore (bottom), fixed with 4% paraformaldehyde, and stained with 50 µg/ml filipin prepared in PBS. (I) Immunoblotting of whole-cell lysates of WT and E-cadherin–KO EpH4 cells with the indicated antibodies. (J) WT and E-cadherin–KO EpH4 cells were fixed with 4% paraformaldehyde and stained with 50 µg/ml filipin prepared in PBS. (K) E-cadherin–KO EpH4 cells were treated with PBS (control) or 75 mM cholesterol-saturated MβCD, fixed, and stained with an anti–claudin-3 pAb. Bars: (A, B, F–H, J, and K) 20 µm; (E) 200 nm.