Vinculin is a component of AJs in FLSs. Indirect immunofluorescence was conducted on FLSs that were cultured for 3 days on coverslips using phalloidin (green) to label F-actin, an antiserum to α-catenin (green), or an antibody to vinculin (red). A: The antibody to vinculin (red) labeled focal contacts (short arrows) as well as AJs (long arrow points to adhesion zipper with multiple clusters of AJs labeled as short parallel lines). B: The antiserum to α-catenin labeled AJs but not focal contacts. Co-localization of vinculin and α-catenin at AJs resulted in yellow labeling at sites of cell-to-cell contact in the merged picture (long arrow).

Cadherin-11-stimulated cell spreading of FLSs is accompanied by clustering of AJs. FLSs were seeded on cadherin-11-Fc- or fibronectin-coated coverslips (10 μg/ml, respectively) and incubated for 90 minutes followed by indirect immunofluorescence using antibodies as indicated. Phalloidin staining revealed lamellipodia (top, short arrow), radial actin fibers (top, long arrows), and circumferential actin fibers (top). The antibody to β-catenin labeled lamellipodia (top, short arrow) and clusters of AJs (top, long arrows) at the cell periphery of FLSs seeded on cadherin-11-Fc-coated coverslips. Note radial actin fibers emanating from clusters of AJs. The α-catenin antiserum and the antibody to p120-catenin both detected clusters of AJs at the cell periphery similar to the β-catenin antibody (middle, long arrows). In FLSs plated on fibronectin, the antibody to paxillin labeled focal contacts that were not detected by the α-catenin antiserum (bottom, long arrows).

Cadherin-11 localization to cell-to-cell junctions. A: FLSs were dispersed in collagen (Vitrogen) and cultured for 5 days. Thereafter, the three-dimensional structure was fixed with 2% paraformaldehyde, permeabilized with 0.2% saponin, and stained with phalloidin (green), the 5H6 antibody to cadherin-11 (red), and DRAQ5 to label nuclei (blue). The FLSs were analyzed by confocal microscopy. Several z-sections were rendered together. Consistent with AJ formation, specific cadherin-11 reactivity was detected at sites of cell-to-cell contact (area in white box is magnified in inset). From AJs, actin fibers radiated internally along the longitudinal axis of the FLS. Note the condensed network of cells forming a lining structure at the interface between the matrix and the fluid phase. B: The isotype control antibody did not detect AJs at sites of cell-to-cell contact. Scale bars, 20 μm.

Cadherin-11 determines lining formation and matrix organization in synovial organ cultures. A and B: Cadherin-11-Fc disrupts FLS lining layer formation. FLSs were released from the culture dish and preincubated with cadherin-11-Fc fusion protein, control human IgG1, or E-cadherin-Fc fusion protein (100 μg/ml, respectively) for 15 minutes at 4°C. Cells and proteins were then dispersed in Matrigel (A) or Vitrogen (B), and cultured for 10 to 12 days in the presence of cadherin-11-Fc, human IgG1, or E-cadherin-Fc (100 μg/ml, respectively). FLSs treated with the cadherin-11-Fc fusion protein failed to establish lining architecture, whereas E-cadherin-Fc or IgG1 treatment had no effect on lining layer formation. C: Cadherin-11 determines organization of reticular fibers. Synovial organ cultures treated with either E-cadherin-Fc fusion protein or cadherin-11-Fc fusion protein (see above) were processed for histochemical analysis using the Gomori silver staining technique to label reticular fibers and nuclear fast red to label FLSs. Note orientation of reticular fibers (black linear structures, arrowspointing to examples) along the FLS lining layer at the tissue interface in E-cadherin-Fc-treated three-dimensional FLS organ cultures. In the sublining area, reticular fibers could be detected as well (irregular, black linear structures, often associated with nuclear fast red-labeled FLSs, arrowspointing to examples). By contrast, in cadherin-11-Fc-treated three-dimensional FLS organ cultures a more chaotic pattern of reticular fibers (short black linear structures throughout the tissue, arrowspointing to examples) could be observed. Specifically, a particular orientation of reticular fibers at the tissue interface could not be seen. Original magnifications: ×200 (A, B); ×400 (C).

Formation of cadherin-11-mediated AJs in FLSs. A: Cadherin-11 localizes to cell-to-cell contacts in cultured FLSs. FLSs were cultured on coverslips for 3 days and processed for indirect immunofluorescence microscopy using phalloidin to label F-actin (green) and anti-cadherin-11 (3H10) to label AJs. At sites of cell-to-cell contact, FLSs demonstrated numerous filopodia-like processes that interdigitated with filopodia from neighboring cells to form an adhesion zipper (area in white box is magnified in inset). F-Actin fibers radiated internally from intercellular junctions (green). The antibody to cadherin-11 labeled clusters of AJs as short parallel lines (red). By contrast, an isotype control antibody did not label AJs. B: Formation of cadherin-11-mediated AJs in FLSs. Western blot detection of cadherin-11, β-catenin, α-catenin, and p120-catenin from FLS immunoprecipitates. Confluent FLSs were lysed with Brij 97. Components of AJs were immunoprecipitated with antibodies against cadherin-11 (3H10), β-catenin, or p120-catenin. An isotype antibody against an unknown antigen was used as a control. β-Catenin, α-catenin, and p120-catenin co-immunoprecipitated with cadherin-11. Multiple p120-catenin species co-immunoprecipitated with cadherin-11 represent known isoforms of p120-catenin. Cadherin-11, α-catenin, and p120-catenin were detectable in β-catenin precipitates. Cadherin-11, β-catenin, or α-catenin were not detectable in p120-catenin precipitates.

Cadherin-11 engagement stimulates actin polymerization and actin cytoskeletal reorganization. A: FLSs were plated on protein-coated coverslips as indicated in the absence of serum for the indicated times, fixed, permeabilized, stained for F-actin (phalloidin-Alexa 488, green), and analyzed by confocal microscopy. Note time-dependent cell spreading. After 30 minutes the cells plated on cadherin-11-Fc fusion protein demonstrated radial spikes (middle, long arrows) and lamellipodial protrusions (middle, short arrows). After 90 minutes pronounced cell spreading was accompanied by the formation of radial actin fibers (right, long arrows) and circumferential actin fibers (right, short arrows). In contrast, in FLSs plated on poly-l-lysine, radial actin fibers and lamellipodial protrusions were not detectable. B: FLSs were plated on cadherin-11-Fc fusion protein-, fibronectin-, or BSA-coated coverglasses. Cadherin-11-Fc induced pronounced cell spreading and actin fiber formation in a similar way as fibronectin, whereas BSA failed to induce cell spreading. Scale bars, 10 μm.

FLSs establish a synovial lining-like structure when cultured in three-dimensions. A: Schematic representation of the three-dimensional organ culture system. FLSs are dispersed as single cells in liquid ECM and placed on a poly-HEMA-coated culture dish to prevent attachment of the micromass to the culture plastic. The ECM was allowed to solidify at 37°C, overlaid with medium, and cultured as a floating sphere. B: Phase contrast microscopic appearance of FLSs cultured in three-dimensional matrix. Immediately after plating, the FLSs were round shaped and isolated. After 2 days in culture, the cells demonstrated thin cellular extensions (short arrows) and became more elongated. At day 21, the FLSs had developed a spindle-like cell shape (long arrows depict a spindle-shaped FLS embedded within the matrix). C: Lining layer formation at the interface between the matrix and the fluid phase. After 3 weeks in culture, the three-dimensional structure was fixed with 2% paraformaldehyde and embedded in paraffin, and sections were stained with H&E. Strikingly, at the tissue interface, the FLSs established a lining structure one to three cell layers thick. The lining layer was overlaying a sparsely populated ECM. D: Immunohistochemistry on three-dimensional FLS synovial organ cultures. In Matrigel dispersed FLSs were cultured for 28 days. The tissue-like structure was fixed, embedded in paraffin, and sections were processed for immunohistochemistry using antiserum against cadherin-11 or control serum. Cadherin-11-specific reactivity was detected at the lining layer and in cells of the sublining area.

Cadherin-11 expression confers on cells the ability to establish a lining layer at the interface between the matrix and the fluid phase. L-cell transfectants as indicated were dispersed in Matrigel, cultured for 7 days, and processed for histochemistry. H&E-stained sections revealed condensed accumulation of cadherin-11 L cells at the tissue interface and cell aggregates in the sublining area. By contrast, E-cadherin L cells formed tight aggregates but failed to establish a lining structure. Vector control L cells formed loose aggregates without forming a lining layer at the tissue interface. Original magnifications, ×200.