The Cadherin-11 Cytoplasmic Juxtamembrane Domain Promotes -Catenin Turnover at Adherens Junctions and Intercellular Motility

Supplemental Material

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• Movie 1
• Figure 1 - Characterization of L-cells stably transfected with the cadherin-11 ΔCBS construct. (A) In-vitro behavior of cadherin-11 ΔCBS L-cells. After 5 days in culture, cadherin-11 ΔCBS L-cells were loosely organized and failed to aggregate into cell clusters. For comparison with L-cells stably transfected with wild-type cadherin-11 or cadherin-11 ΔJMD see Figure 4. Bar 50 μm. (B) Actin cytoskeletal organization in cadherin-11 ΔCBS L-cells. Cadherin-11 ΔCBS L-cells were plated at intermediate cell density and cultured for 3 days. Then L-cells were processed for fluorescence microscopy using phalloidin-Alexa 488 to label F-actin (green) and the anti-cadherin-11 mAb (3H10) to label AJs (red). Cadherin-11 ΔCBS L-cells demonstrated a pronounced rim of cortical actin at the cell periphery. Few intercellular junctions were labeled with the anti-cadherin-11 mAb. In contrast to wild-type cadherin-11 L-cells or cadherin-11 ΔJMD L-cells, actin fiber organization linked to intercellular junctions such as radial F-actin fibers emanating from intercellular junctions or circular bundles of fibers underlying the intercellular junctions were missing. For comparison see Figure 4B and 4C. (C) Levels of active Rac1 in L-cell transfectants. The levels of Rac1-GTP were measured in confluent L-cells expressing wild-type cadherin-11, mutant cadherin-11, or control vector. Data are representative of four independent experiments. Densitometric analysis was performed using ImageJ software. (D) The Cadherin-11 cytoplasmic domains determine cadherin-11 induced migration. Values are mean ± 1 SD of triplicates (n=3). Cadherin-11 induced migration assays were performed as described in Materials and Methods. (E) Specific adhesion to cadherin-11-Fc. The adhesion assay was conducted as described in Materials and Methods using vector control L-cells, wild-type cadherin-11 L-cells, or mutant cadherin-11 L-cells and microtiter plates directly coated with the indicated concentrations of cadherin-11-Fc protein. Cadherin-11 ΔCBS L-cells demonstrated decreased adhesive capacity when compared to wild-type cadherin-11 L-cells or cadherin-11 ΔJMD L-cells. The results are expressed as the mean percent of cells that were adherent ± 1 SD (n=4).