alpha-catenin-independent recruitment of ZO-1 to nectin-based cell-cell adhesion sites through afadin

Abstract

ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and alpha-catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-catenin complex through alpha-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and alpha-catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only alpha-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of alpha-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with alpha-catenin but also with the nectin-afadin system.

Figure 1

Specificity of antibodies and expression levels of various constructs. The cell lysates of various L cell lines and F9 cell lines (each 20 μg of protein) were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-2 mAb, the anti–nectin-2α pAb, the anti–ZO-1 mAb, the anti–ZO-1 pAb, the anti-afadin mAb, the anti–E-cadherin mAb, or the anti-Myc mAb. (A) EL and nectin-2α-EL cells. Lane 1, EL cells; lane 2, nectin-2α-EL cells. (B) nEα-L, nEαN-L, and nEαC-L cells. Lane 1, nEα-L cells; lane 2, nEαN-L cells; lane 3, nEαC-L cells. (C) Nectin-2α-L and -2α-ΔC-L cells. Lane 1, nectin-2α-L cells; lane 2, nectin-2α-ΔC-L cells. (D) Nectin-2α-L cells transfected with pPGKIZ-Myc-α-catenin or the empty vector. Lane 1, empty vector; lane 2, pPGKIZ-Myc-α-catenin. (E) αF9Dα(−/−) and F9Dα(−/−) cells. Lane 1, αF9Dα(−/−) cells; lane 2, F9Dα(−/−) cells. The results shown are representative of three independent experiments.

Figure 2

Colocalization of ZO-1 with nectin-2α and afadin at E-cadherin–based AJs. Nectin-2α-EL cells were doubly stained with various combinations of the anti–ZO-1 pAb, the anti–nectin-2 mAb, the anti–E-cadherin mAb, and the anti-afadin mAb. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 3

Localization of ZO-1 and nectin-2 at E-cadherin–based AJs through the C-terminal half of α-catenin. nEα-L, nEαN-L, and nEαC-L cells were doubly stained with the anti–ZO-1 mAb and the anti–nectin-2 mAb. (A) nEα-L cells. (B) nEαN-L cells. (C) nEαC-L cells. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 4

Afadin-dependent recruitment of ZO-1 to nectin-2α–based cell-cell adhesion sites. Nectin-2α-L and -2α-ΔC-L cells were doubly stained with various combinations of the anti–ZO-1 mAb, the anti–nectin-2 mAb, and the anti-afadin mAb. (A) Nectin-2α-L cells. (B) Nectin-2α-ΔC-L cells. Arrows, nectin-2α–based cell-cell adhesion sites; arrowheads, nectin-2α-ΔC–based cell-cell adhesion sites. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 5

Afadin-dependent association of ZO-1 with nectin-2α. (A) Subcellular fractionation analysis. Nectin-2α-L and -2α-ΔC-L cells were sonicated, followed by ultracentrifugation. A comparable amount of each fraction (each 20 μg of protein) was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-2α pAb, the anti–ZO-1 mAb, and the anti-afadin mAb. (B) Immunoprecipitation analysis. The cell extracts of nectin-2α-L and -2α-ΔC-L cells (each 2 mg of protein) were separately subjected to immunoprecipitation with the anti–nectin-2 mAb. The immunoprecipitate was then subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-2α pAb, the anti–ZO-1 mAb, and the anti-afadin mAb. Lane 1, nectin-2α-L cells; lane 2, nectin-2α-ΔC-L cells. The results shown are representative of three independent experiments.

Figure 6

Inability of ZO-1 to directly interact with nectin-2α. MBP-ZO-1-PDZ1–2, MBP-ZO-1-PDZ2–3, or MBP-afadin-PDZ (each 20 μg of protein) was immobilized on amylose resin beads. GST-nectin-2α-CP (100 μg of protein) was applied to the affinity beads. After the beads were extensively washed, elution was performed with 10 mM maltose. The eluate was subjected to SDS-PAGE (13% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. The results shown are representative of three independent experiments.

Figure 7

Afadin-dependent corecruitment of ZO-1 and α-catenin to nectin-2α–based cell-cell adhesion sites. Nectin-2α-L and -2α-ΔC-L cells were transfected with pPGKIZ-Myc-α-catenin and triply stained with the anti-Myc mAb, the anti–ZO-1 pAb, and the anti–nectin-2 mAb. (A) Nectin-2α-L cells. (B) Nectin-2α-ΔC-L cells. Arrows, nectin-2α–based cell-cell adhesion sites; arrowheads, nectin-2α-ΔC–based cell-cell adhesion sites. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 8

α-Catenin-independent colocalization of ZO-1 with nectin-2 and afadin. αF9Dα(−/−) and F9Dα(−/−) cells were doubly stained with various combinations of the anti–ZO-1 mAb, the anti–nectin-2 mAb, the anti–E-cadherin mAb, and the anti-afadin mAb. (A) αF9Dα(−/−) cells. (B) F9Dα(−/−) cells. Arrows, cell-cell adhesion sites between two cells; arrowheads, cell-cell adhesion sites where more than two cells adhere to each other. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 8

α-Catenin-independent colocalization of ZO-1 with nectin-2 and afadin. αF9Dα(−/−) and F9Dα(−/−) cells were doubly stained with various combinations of the anti–ZO-1 mAb, the anti–nectin-2 mAb, the anti–E-cadherin mAb, and the anti-afadin mAb. (A) αF9Dα(−/−) cells. (B) F9Dα(−/−) cells. Arrows, cell-cell adhesion sites between two cells; arrowheads, cell-cell adhesion sites where more than two cells adhere to each other. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 9

Recruitment of ZO-1 to nectin-2α–based cell-cell adhesion sites in a manner insensitive to latrunculin A. Nectin-2α-L cells were incubated in the presence or absence of 50 nM latrunculin A for 45 min, followed by double staining with various combinations of rhodamine-phalloidin, the anti–nectin-2 mAb, the anti-afadin mAb, and the anti–ZO-1 mAb. (A) F-actin and nectin-2. (B) Afadin and nectin-2. (C) ZO-1 and nectin-2. Arrows, nectin-2α–based cell-cell adhesion sites. Bars, 10 μm. The results shown are representative of three independent experiments.

Figure 9

Recruitment of ZO-1 to nectin-2α–based cell-cell adhesion sites in a manner insensitive to latrunculin A. Nectin-2α-L cells were incubated in the presence or absence of 50 nM latrunculin A for 45 min, followed by double staining with various combinations of rhodamine-phalloidin, the anti–nectin-2 mAb, the anti-afadin mAb, and the anti–ZO-1 mAb. (A) F-actin and nectin-2. (B) Afadin and nectin-2. (C) ZO-1 and nectin-2. Arrows, nectin-2α–based cell-cell adhesion sites. Bars, 10 μm. The results shown are representative of three independent experiments.