Junctional organization in DLD-1, R2/7, and R2/7-αE cells. (A) Immunoblot analysis for E-cadherin (lanes 1 and 2), β-catenin (lanes 3 and 4), and αE-catenin (lanes 5 and 6) in DLD-1 (lanes 1, 3, and 5) or in DLD-1/R2/7 (lanes 2, 4, and 6). Molecular weight markers are 200, 116, 97, 66, and 45 × 10³. (B–D) Phase-contrast micrographs of DLD-1 (B), R2/7 (C), and R2/7-αE (D). (E–J) Confocal images of double immunofluorescence staining for ZO-1 (E–G) and E-cadherin (H–J). (E, H) R2/7-αE. The focus was adjusted to an apical plane. (F, G, I, and J) R2/7. The focus was adjusted to two planes: apical (F, I) and middle (G, J). (K–T) Immunofluorescence staining for junctional or cytoskeletal proteins in R2/ 7-αE (K–O) and R2/7 (P–T). The focus was adjusted to their apical planes. Cells were double-stained for ZO-1/F-actin in K and L and in P and Q, and were singly stained for occludin (M, R), α-actinin (N, S), or vinculin (O, T). (U, V) Freeze-fracture replica views of R2/7-αE (U) and R2/7 (V) at junctional areas. TJ strands (arrows) are detected not only in U but also in V. zo, ZO-1; cad, E-cadherin; act, actin; occ, occludin; αac, α-actinin; vin, vinculin. These abbreviations are also used in other figures. Bars: (B–D) 100 μm; (E–J and K–T) 20 μm; (U and V) 200 nm.

Association pattern of cells expressing various αE-catenin constructs. (A–J) Vertical EM sections of cells in monolayer cultures seen at a low magnification (A–C, G and H), and their close-up views at apical cell–cell junction areas (D–F, I and J). (A and D) R2/7-αE. (B and E) R2/7-αE(1–509). (C and F) R2/7-αE/ vinTail. (G and I) R2/7. (H and J) R2/7-αE(1–325/510–890). Insert in I is another section of R2/7, showing a desmosome at the apical-most junction. Arrows in B point to a boundary between two cells that irregularly overlap each other. Asterisks indicate TJ-ZA complexes. Arrowheads in I point to filopodial contacts with electron-dense cytoplasmic materials. d, desmosome. (K–Q) Aggregates of R2/7 cells and their αE-catenin transfectants in the absence (K–O) or presence (P and Q) of anti-E-cadherin antibodies. (K and P) R2/7-αE. (L and Q) R2/7. (M) R2/7-αE(1– 509). (N) R2/7-αE(1–325/510–890). (O) R2/7-αE/vinTail. Cells were trypsinized into single cells and cultured overnight in agar-coated dishes. Bars: (A–C, G, and H) 2 μm; (D–F, I and J) 200 nm; (K–Q) 20 μm.

Only αE(1–509) can rescue R2/7 cells to organize the TJ. (A, D, and G) R2/7-αE(1–509). (B, E, and H) R2/7-αE(1–325). (C, F, and I) R2/7-αE(1–325/510–890). (A–C) Phase-contrast micrographs of cells expressing these mutant αE-catenins. (D–I) Confocal images of double immunofluorescence staining for ZO-1 (D–F) and E-cadherin (G–I). Bars: (A–C) 100 μm; (D–I) 20 μm.

An αE-catenin/vinculin chimeric protein can redistribute ZO-1. (A) Schematic drawing of αE-catenin/vinculin chimeric proteins. The NH₂-terminal 1–325 amino acid domain was fused with the NH₂-terminal 1–823 or COOH-terminal 822–1067 domain of vinculin. (B and D) Double immunofluorescence staining for ZO-1 (B) and T7 tag (D) of cells expressing αE/vinTail. (C and E) Double immunofluorescence staining for ZO-1 (C) and T7 tag (E) of cells expressing αE/vinHead. (F and G) Double immunofluorescence staining for E-cadherin (F) and α-actinin (G) in cells expressing αE/vinTail. Staining signals observed in the nuclei are due to nonspecific association of the anti-T7 tag antibodies with these structures. Bars (B–E; F and G), 10 μm.

Colocalization of vinculin or α-actinin with E-cadherin. (A and D) R2/7; (B and E) R2/7-αE; (C and F; H and K) R2/7-αE(1–509). (G and J; I and L) R2/7-αE(1–325/510–890). Double immunofluorescence staining for E-cadherin and vinculin (A and D; B and E; C and F; G and J), and for E-cadherin and α-actinin (H and K; I and L). A–G and J are confocal images. Bars: (A–G and J; H, I, K, and L) 20 μm.

Deletion constructs of αE-catenin and their characterization. (A) Schematic drawing of the mutant αE-catenin constructs, designated as 1–8, introduced into R2/7 cells. Stable transfectants were isolated for each construct. (B) Immunoblot detection of the mutant αE-catenin proteins (1–8) expressed by the transfectants with anti-T7 tag antibodies. (C) Immunoblot detection of E-cadherin coimmunoprecipitated with the mutant αE-catenin molecules. Materials immunoprecipitated with an anti-T7 tag antibody from a lysate of each transfectant were subjected to detection of E-cadherin. The numbers marking the lanes in B and C correspond to the construct number in A. Lower bands seen in some lanes are likely degradation products of the original molecule. Positions of molecular markers are the same as in Fig. 1 A.

In vitro binding of vinculin with αE-catenin. (A) Schematic drawing of GST-αE-catenin fusion proteins (1–7), and of MBP-vinculin fusion proteins (MBP-vinHead and MBP-vinTail). (B) Detection of proteins bound to the GST-αE-catenin fusion proteins 2, 3, and 7 (control) that had been incubated with chicken gizzard extracts. (Top) proteins bound to the GST-fusion proteins were separated by SDS-PAGE, and were visualized by silver staining. Two bands of 130 and 120 kD (arrowheads) were precipitated with GST-αE(1–509) (2), but not with the other constructs. These bands were recognized with anti-vinculin antibodies (middle). α-Actinin and spectrin did not bind to any of these constructs, whereas β-catenin was precipitated with 2 as well as with 3 (bottom). giz, the original extract of gizzards used for these binding assays; vin, vinculin; αac, α-actinin; spe, spectrin; βca, β-catenin. Positions of molecular markers are 200, 116, and 97 × 10³. (C) Binding of MBP-vinHead (vinH) or MBP-vinTail (vinT) to GST-αE-catenin fusion proteins 1–7. Sepharose beads conjugated with these GST-fusion proteins were incubated with a lysate of E. coli expressing the MBP-fusion proteins. Coprecipitated proteins were analyzed by Western blotting with antibodies to vinculin (for MBP-vinHead) or to MBP (for MBP-vinTail; top). Each sample for electrophoresis contained an equal molar amount of the GST-fusion proteins that had been adjusted before loading on the gel. (Bottom) Coomassie blue staining for the GST-αE-catenin fusion proteins 1–7 conjugated to Sepharose beads. Positions of molecular markers are the same as in Fig. 1 A. lys, the original lysate of E. coli used for this binding assay.

Junctional organization in normal and vinculin-null F9 cells. (A–H) Low cell density. Cells (1 × 10⁵) were inoculated on a collagen-coated coverslip placed in each 3.5-cm dish, and were cultured for 2 d. (I–R) High cell density. The above cultures were maintained for 4 d. (A, B, E, F, I, J, M, and N) Normal F9 cells. (C, D, G, H, K, L, O, and P) Vinculin-null γ229 cells. (Q and R) R15 cells that were isolated by transfection of γ229 with vinculin cDNA. ZO-1 was double-stained with vinculin (A and B; C and D; I and J; K and L; Q and R), and with E-cadherin (E and F; G and H; M and N; O and P). Note the web-like organization of ZO-1 in vinculin-positive cells cultured for 4 d. Such ZO-1 organization is prohibited in the absence of vinculin. Confocal images focused at the apical-most plane of cells are presented. zo, ZO-1; cad, E-cadherin; vin, vinculin. Bar, 20 μm.