Tight junction formation by a claudin mutant lacking the COOH-terminal PDZ domain-binding motif

Abstract

Claudin-based tight junctions (TJs) are formed at the most apical part of cell-cell contacts in epithelial cells. Previous studies suggest that scaffolding proteins ZO-1 and ZO-2 (ZO proteins) determine the location of TJs by interacting with claudins, but this idea is not conclusive. To address the role of the ZO proteins binding to claudins at TJs, a COOH-terminal PDZ domain binding motif-deleted claudin-3 mutant, which lacks the ZO protein binding, was stably expressed in claudin-deficient MDCK cells. The COOH-terminus-deleted claudin-3 was localized at the apicolateral region similar to full-length claudin-3. Consistently, freeze-fracture electron microscopy revealed that the COOH-terminus-deleted claudin-3-expressing cells reconstituted belts of TJs at the most apical region of the lateral membrane and restored functional epithelial barriers. These results suggest that the interaction of claudins with ZO proteins is not a prerequisite for TJ formation at the most apical part of cell-cell contacts.

Keywords: PDZ domain-binding motif; ZO-1; claudin; tight junction.

FIGURE 1

Expression and localization of exogenous claudin‐3 and claudin‐3ΔYVF in claudin quinKO cells. (A) Western blots of claudin quinKO cells, two clones of claudin‐3‐expressing cells, and two clones of claudin‐3ΔYVF‐expressing cells with anti‐claudin‐3 antibody, anti‐FLAG antibody, or anti‐α‐tubulin antibody. (B) (Top panel) Immunofluorescence staining of claudin quinKO cells showed claudin‐3 is absent, while ZO‐1 and occludin strictly localize at apical cell–cell contacts. (Middle panel) Claudin‐3‐expressing cells clone 2 (+ Claudin‐3) double‐stained with anti‐claudin‐3 and anti‐ZO‐1 or anti‐claudin‐3 and anti‐occludin. Claudin‐3 is present along the lateral membrane and colocalized with ZO‐1 and occludin at apical cell–cell contacts. (Bottom panel) Claudin‐3ΔYVF‐expressing cells clone 2 (+ Claudin‐3ΔYVF) double‐stained with anti‐FLAG and anti‐ZO‐1 or anti‐FLAG and anti‐occludin antibodies. FLAG signals, which represent claudin‐3ΔYVF, located along the lateral membrane and colocalized with ZO‐1 and occludin at apical cell–cell contacts. The orthogonal views are shown below each image for the z‐stack distribution; a and b with arrows represent apical and basal, respectively. Scale bar 20 μm.

FIGURE 2

TJ strands reconstituted in claudin‐3‐expressing cells. (A, B) Two independent freeze‐fracture replica images of claudin‐3‐expressing cells. Continuous belts of TJ strands are observed at the most apical region of the lateral membrane (arrows). MV, microvilli. Scale bar: 500 nm.

FIGURE 3

TJ strands reconstituted in claudin‐3ΔYVF‐expressing cells. (A) Freeze‐fracture replica of claudin‐3ΔYVF‐expressing cells. (A') TJ strands in (A) are traced in white. Dotted rectangles (b), (c), and (d) are enlarged in (B), (C), and (D), respectively to clearly show TJ strands. TJ strands are reconstituted at the most apical region of the lateral membrane close to microvilli (MV) (B, C). (C) Fragmented TJ strands are observed in the lateral membrane (arrowheads). (D) Discontinuity of apical TJ strands (arrow). Scale bars: 500 nm in A'; 200 nm in B and C; 100 nm in D.

FIGURE 4

Epithelial barrier function of claudin‐3‐ and claudin‐3ΔYVF‐expressing cells. (A) TER measurements of claudin quinKO cells, claudin‐3‐expressing cells, and claudin‐3ΔYVF‐expressing cells. Expression of claudin‐3 or claudin‐3ΔYVF increased the TER value. The effect of claudin‐3 was greater compared to claudin‐3ΔYVF. N = 3 each, unpaired, two‐tailed t‐test. (B) The paracellular flux of fluorescein in claudin quinKO cells, claudin‐3‐expressing cells, and claudin‐3ΔYVF‐expressing cells. Expression of claudin‐3 or claudin‐3ΔYVF reduced the paracellular diffusion of fluorescein. The effect of claudin‐3 was greater compared to claudin‐3ΔYVF. The right panel is an enlargement of the dotted box region in the left graph. N = 3 each, unpaired, two‐tailed t‐test.

FIGURE 5

Actomyosin organization of claudin‐3‐expressing cells and claudin‐3ΔYVF‐expressing cells. Visualization of ZO‐1, F‐actin, and myosin IIB in MDCK II cells, claudin quinKO cells, claudin‐3‐expressing cells clone 2, and claudin‐3ΔYVF‐expressing cells clone 2. F‐actin and myosin IIB were clearly concentrated at the cell junctions in claudin quinKO cells, but not in MDCK II cells, claudin‐3‐expressing cells, or claudin‐3ΔYVF‐expressing cells. Scale bar, 10 μm.

FIGURE 6

Interaction between ZO‐1 and claudin‐3. HEK293 cells cotransfected with ZO‐1‐GFP and claudin‐3 or claudin‐3ΔYVF were lysed and immunoprecipitated with an anti‐GFP antibody. Immunoprecipitates (IP) and lysates were analyzed by immunoblotting with an anti‐HA or an anti‐GFP antibody. ZO‐1 coprecipitated with claudin‐3 but not with claudin‐3ΔYVF. IB, immunoblot.