Direct binding of l-afadin to nectin in vitro. (A) Affinity chromatography. GST-l-afadin-PDZ (aa 1007–1125) or GST alone (20 μg of protein each) was applied to protein A–Sepharose beads on which Myc-nectin-1-CP (aa 379–518) was immobilized through the anti-Myc mAb. After the beads were extensively washed, the bound proteins were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. Arrow, GST-l-afadin-PDZ; arrowhead, Myc-nectin-1-CP. (B) ³⁵S-Labeled l-afadin blot overlay. GST-fusion proteins of the cytoplasmic regions of nectin (0.3 μg of protein each) were subjected to SDS-PAGE (12% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue or by ³⁵S-labeled l-afadin blot overlay. (B1) Protein staining. (B2) ³⁵S-Labeled l-afadin blot overlay. GST-Nectin-1-CPC, aa 449–518; GST-Nectin-1-CPC-ΔC, aa 449–514; GST-Nectin-2α-CP, aa 387–467; GST-Nectin-2α-CP-ΔC, aa 387–463; GST-Nectin-2δ-CP, aa 403–530; GST-Nectin-2δ-CP-ΔC, aa 403–526.

Localization of nectin, l-afadin, and E-cadherin in epithelial cells. (A) Localization sites of nectin-2, l-afadin, and E-cadherin in mouse small intestine. The frozen sections of mouse small intestine were triply stained with the rat anti–nectin-2 mAb, the rabbit anti–l-afadin pAb, and the mouse anti–E-cadherin mAb. They were visualized with rhodamine-conjugated anti– rat IgG, FITC-conjugated anti– rabbit IgG, and Cy5-conjugated anti–mouse IgG Abs. (A1) Nectin-2; (A2) l-afadin; (A3) E-cadherin. Asterisks mark the inner space of small intestine. Bar, 50 μm. (B) Localization sites of nectin-1, l-afadin, and E-cadherin in MDCK cells. MDCK cells stably expressing FLAG-nectin-1 were triply stained with the mouse anti-FLAG mAb, the rabbit anti–l-afadin pAb, and the rat anti–E-cadherin mAb. They were visualized with FITC-conjugated anti–mouse IgG, rhodamine-conjugated anti–rabbit IgG, and Cy5-conjugated anti–rat IgG Abs. (B1 and B4) FLAG-nectin-1; (B2 and B5) l-afadin; (B3 and B6) E-cadherin; (B1–B3) junction-level view; (B4–B6) cross-sectional view. The cross-sectional view was generated by confocal microscopy. Arrow, apical level; arrowhead, basal level. Bars, 10 μm.

Localization of nectin and l-afadin in nonepithelial cells. (A) Localization sites of nectin-2 and l-afadin in mouse heart. The frozen sections of mouse heart were doubly stained with the rat anti–nectin-2 mAb and the rabbit anti–l-afadin pAb. They were visualized with rhodamine-conjugated anti–rat IgG and FITC-conjugated anti–rabbit IgG Abs. (A1) Nectin-2; (A2) l-afadin. Arrows, intercalated disc. Bar, 10 μm. (B) Localization sites of nectin-2 and l-afadin in cultured EL cells. EL cells were doubly stained with the rat anti–nectin-2 mAb and the mouse anti–l-afadin mAb. They were visualized with FITC-conjugated anti–rat IgG and rhodamine-conjugated anti–mouse IgG Abs. There was nuclear staining with this anti–l-afadin mAb, but its significance is not clear. (B1) Nectin-2; (B2) l-afadin. Bar, 10 μm.

Cell aggregation activity of nectin. (A) Ca²⁺-independent aggregation activity of nectin-1 and -2α. L cells stably expressing nectin-1 or nectin-2α were treated with trypsin in the presence of EDTA and then dispersed by pipetting to obtain a single-cell suspension. Each single-cell suspension was rotated in HBSS in the presence of 1 mM CaCl₂ or 1 mM EDTA for 15, 30, and 60 min. The extent of aggregation of cells was represented by the ratio of the total particle number at time t of incubation (Nt) to the initial particle number (No). (A1) Nectin-1-L cells. (Filled circles and open circles) Wild-type L cells; (filled triangles and open triangles) nectin-1-L cells; (filled circles and filled triangles) in the presence of 1 mM CaCl₂; and (open circles and open triangles) in the presence of 1 mM EDTA. (A2) Nectin-2α-L cells. (Filled circles and open circles) Wild-type L cells; (filled triangles and open triangles) nectin-2α-L cells; (filled circles and filled triangles) in the presence of 1 mM CaCl₂; and (open circles and open triangles) in the presence of 1 mM EDTA. The Nt/No values are the means ± SD of three independent experiments. (B) Cell aggregation of nectin-1-L cells. Single cells were rotated in HBSS in the presence of 1 mM EDTA for 60 min. (B1) Wild-type L cells. (B2) Nectin-1-L cells. Bars, 100 μm. These results are representative of three independent experiments.

Structures of the full length and various fragments of l-afadin and nectin. PR, proline-rich region; ABD, F-actin–binding domain; TM, transmembrane region.

Coimmunoprecipitation of l-afadin and nectin. (A) Immunoprecipitation from the bile canaliculi-rich fraction of mouse liver. An extract from the bile canaliculi-rich fraction was subjected to immunoprecipitation with the anti–nectin-2 mAb or control IgG. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti–l-afadin mAb or the anti–nectin-2α pAb. (B) Immunoprecipitation from mouse mammary tumor MTD-1A cells. A cell extract from MTD-1A cells was subjected to immunoprecipitation with the anti–nectin-2 mAb or control IgG. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti–l-afadin mAb, the anti–nectin-2α pAb, or the anti– nectin-2δ pAb. (C) Immunoprecipitation from COS7 cells expressing FLAG-nectin-1 and/or Myc-l-afadin. FLAG-Nectin-1 alone, Myc-l-afadin alone, or both the proteins were transiently expressed in COS7 cells. Each cell extract was subjected to immunoprecipitation with the anti-FLAG or anti-Myc mAb. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti-FLAG or anti-Myc mAb. IP, immunoprecipitation.

Ultrastructural localization sites of nectin-2 in mouse small intestine absorptive epithelial cells. Intestine absorptive epithelial cells were labeled with the anti–nectin-2 mAb using the silver-enhancement technique. Open arrow, ZO; closed arrow, ZA; asterisk, desmosome. Bars, 0.2 μm.

Recruitment of nectin to cadherin-based AJs through interaction with l-afadin. (A) Immunoprecipitation from EL cells expressing FLAG-nectin-1 or FLAG-nectin-1-ΔC. FLAG-Nectin-1 or FLAG-nectin-1-ΔC was transiently expressed in EL cells. Each cell extract was subjected to immunoprecipitation with the anti-FLAG mAb. The immunoprecipitate was then subjected to SDS-PAGE (8 or 12% polyacrylamide gel), followed by Western blot analysis with the anti-FLAG or anti–l-afadin mAb. (B) Immunofluorescence microscopy of FLAG-nectin-1-EL cells and FLAG-nectin-1-ΔC-EL cells. FLAG-nectin-1-EL cells and FLAG-nectin-1-ΔC-EL cells were doubly stained with the mouse anti-FLAG mAb and the rat anti–E-cadherin mAb or the rabbit anti–l-afadin pAb. They were visualized with FITC-conjugated anti–mouse IgG and rhodamine-conjugated anti–rat or anti–rabbit IgG Abs. There was nuclear staining with this anti–l-afadin pAb, but its significance is not clear. (B1–B6) FLAG-nectin-1-EL cells; (B7–B12) FLAG-nectin-1-ΔC-EL cells; (B1 and B4) FLAG-nectin-1; (B7 and B10) FLAG-nectin-1-ΔC; (B2 and B8) E-cadherin; (B5 and B11) l-afadin; (B3, B6, B9, and B12) merge. Arrows, cell–cell AJs. Bars, 10 μm.