Involvement of the interaction of afadin with ZO-1 in the formation of tight junctions in Madin-Darby canine kidney cells

Abstract

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta- and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-DeltaPR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

FIGURE 1.

Association of afadin with ZO-1 in cells cultured at a low Ca²⁺ concentration. A, immunofluorescence staining for ZO-1, afadin, and E-cadherin in MDCK cells. MDCK cells cultured in the presence of 2 mm Ca²⁺ (Normal Ca²⁺) were precultured at 2 μm Ca²⁺ for 3 h (Low Ca²⁺). The cells were then re-cultured in the presence of 2 mm Ca²⁺ for 4 h (Ca²⁺ switch) or cultured at 2 μm Ca²⁺ containing 100 nm TPA for 1 h (Low Ca²⁺+TPA). Cells under each condition were triple stained with the anti-ZO-1, anti-afadin, and anti-E-cadherin Abs. Arrowheads indicate the localization of ZO-1, which was separate from those of afadin and E-cadherin, and an arrow indicates the co-localization of ZO-1 and afadin. Scale bars, 10 μm. B, binding of afadin and ZO-1 in MDCK cells. Cell extracts of MDCK cells cultured under each condition were immunoprecipitated with the anti-afadin mAb and subjected to Western blotting using the anti-afadin and anti-ZO-1 Abs. IP, immunoprecipitation; N, Normal Ca²⁺; L, Low Ca²⁺; L+TPA, Low Ca²⁺+TPA; CS, Ca²⁺ switch.

FIGURE 2.

Requirement of ZO-1 for the formation of TJs, but not AJs. A, expression levels of AJ and TJ markers in ZO-1-knockdown MDCK cells. Cell extracts of control and ZO-1-knockdown MDCK cells were separated by SDS-PAGE and subjected to Western blotting using the indicated Abs. Actin was immunoblotted as a loading control. B–D, delay in the assembly of the immunofluorescence signal for occludin, but not afadin or E-cadherin, at cell-cell adhesion sites in ZO-1-knockdown MDCK cells. At the indicated time points after the Ca²⁺ switch, control and ZO-1-knockdown MDCK cells were fixed and stained with the indicated Abs. Arrowheads indicate the absence of the signal for occludin. Scale bars, 10 μm.

FIGURE 2.

Requirement of ZO-1 for the formation of TJs, but not AJs. A, expression levels of AJ and TJ markers in ZO-1-knockdown MDCK cells. Cell extracts of control and ZO-1-knockdown MDCK cells were separated by SDS-PAGE and subjected to Western blotting using the indicated Abs. Actin was immunoblotted as a loading control. B–D, delay in the assembly of the immunofluorescence signal for occludin, but not afadin or E-cadherin, at cell-cell adhesion sites in ZO-1-knockdown MDCK cells. At the indicated time points after the Ca²⁺ switch, control and ZO-1-knockdown MDCK cells were fixed and stained with the indicated Abs. Arrowheads indicate the absence of the signal for occludin. Scale bars, 10 μm.

FIGURE 3.

The ZO-1-binding region of afadin. A, schematic diagram of afadin fragments. Afadin fragments were tagged with a FLAG or HA epitope at the N terminus. +, positive for the interaction with ZO-1; −, negative for the interaction with ZO-1. B and C, co-immunoprecipitation of ZO-1 with the PR1–2 region of afadin. Cell extracts of HEK293 cells transfected with various combinations of the indicated vectors were immunoprecipitated with the anti-FLAG or anti-HA mAb. Immunoprecipitates and cell extracts were subjected to Western blotting using the anti-FLAG and anti-HA mAbs.

FIGURE 4.

The afadin-binding region of ZO-1. A, schematic diagram of ZO-1 fragments. All ZO-1 fragments were tagged with an HA epitope at the N terminus. +, positive for the interaction with afadin; −, negative for the interaction with afadin. B–D, co-immunoprecipitation of afadin with the SH3 domain of ZO-1. The immunoprecipitation experiment using HEK293 cells transfected with various combinations of the indicated vectors was performed as described in the legend to Fig. 3.

FIGURE 5.

Requirement of the interaction of afadin with ZO-1 for the formation of TJs. Discontinuous localization of TJ components at cell-cell adhesion sites in afadin-knockdown MDCK cells re-expressing GFP-afadin-ΔPR1–2. After the Ca²⁺ switch, MDCK cells were fixed and stained in the combination with the anti-ZO-1 mAb and anti-afadin pAb, the anti-JAM-A pAb and anti-afadin mAb, or the anti-E-cadherin mAb and anti-afadin pAb. A, afadin-knockdown MDCK cells expressing GFP. B, afadin-knockdown MDCK cells re-expressing GFP-afadin. C, afadin-knockdown MDCK cells re-expressing GFP-afadin-ΔPR1–2. Arrowheads indicate the absence of the signals for ZO-1, JAM-A, or E-cadherin. Scale bars, 10 μm. D, bars in the graph represent the relative immunofluorescence intensities of ZO-1, JAM-A, and E-cadherin at the interface between GFP-, GFP-afadin-, or GFP-afadinΔPR1–2-expressing cells as compared with the values at the interface between GFP-negative cells, which are expressed as 1. E, paracellular diffusion of FITC-dextran (average 40 kDa) in afadin-knockdown MDCK cells re-expressing GFP, GFP-afadin, or GFP-afadin-ΔPR1–2. Error bars in D and E indicate S.E. **, p < 0.01.