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Fig. 2. Shroom2 binds to F actin. (A,B) Using (A) the anti-mShrm2R1 antibody, faint shroom2 labeling is observed in some MDCK cells, especially at cell-cell contacts. Shroom2 colocalizes with ZO-1 (yellow in right panel) especially at tricellular contacts. In (B) transfected MDCK cells that express GFP-labeled full-length shroom2 (GFP-mShrm2) a similar distribution pattern is displayed. Both endogenous shroom2 (A) and GFP-mShm2 (B) labeling is more intense at tricellular contacts (arrowheads in A and B). The TJs are visualized by ZO-1 labeling (red in A,B). (C,D) In MDCK cells expressing GFP-mShrm2 that were treated with cytochalasin D, the GFP-mShrm2 (green) colocalizes with F-actin (red), along the cell membrane. (E,F) By contrast, in cells expressing GFP-mShrm2MBR (green) that were treated with cytochalasin D, the large F-actin bundles induced by overexpression of GFP-mShrm2MBR (red) are protected against depolymerization (arrowheads). (G) In MDCK cells that express GFP alone, a diffuse GFP staining is observed in cytochalasin-D-treated cells. (H) F-actin co-sedimentation assay. Lanes 1 and 2 contain the soluble and pellet fractions of GST-mShrm2MBR, respectively, obtained upon high-speed centrifugation. GST-mShrm2MBR is not recovered in the pellet fraction (lane 2). When the same amount of GST-mShrm2MBR is incubated with F-actin at 37°C for 30 minutes (lanes 3 and 4), almost all GST-mShrm2MBR is recovered with F-actin in the pellet fraction after ultracentrifugation (lane 4). GST alone was used as a negative control (lanes 5 and 6). Bars, 20 µm.
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