Figure 9.
Down-regulation of 4.1R+17b expression affects the organization of the spectrin–actin skeleton at cell–cell contacts and attenuates the initial reassembly of E-cadherin at the AJs. A–C, MDCK Tet-On stable lines were generated with a control scramble shRNA (Ctrl) or 4.1R-exon 17b shRNA (sh17b) in pTRIPZ where RFP served as an expression marker. sh17b lines grown in the absence (−dox) or presence (+dox) of doxycycline (dox) and sh17b line co-expressing rescue construct 4.1R135+17b grown in the presence of doxycycline (+dox 4.1Rres) were analyzed. A, 4.1R+17b is silenced at the intercellular junctions, and 4.1R135+17b rescue restores its expression at the cell–cell contacts. sh17b lines with indicated treatments were immunofluorescently-stained with an anti-ex17b antibody and revealed with a Zeiss microscope. Bar, 10 μm. B, 4.1R+17b silence and rescue do not affect the protein levels of the AJs and spectrin–actin cytoskeleton. Western blotting of cell lysates from control and sh17b cell lines treated as indicated were analyzed for ex17b, 4.1R rescue forms (Flag), E-cadherin, β-catenin, α-spectrin, βII-spectrin, and β-actin with its respective antibody. GAPDH served as a loading control. Molecular mass markers (kDa) are provided. C, immunofluorescence staining for actin and βII-spectrin in sh17b cells treated as indicated (+dox and +dox 4.1Rres) and revealed with a Zeiss microscope. Upper panel, depletion of 4.1R+17b forms results in the reduction of actin at the AJs, and 4.1R135+17b expression restores cell–cell contact localization of actin. Lower panel, βII-spectrin in 4.1R+17b-depleted cells largely distribute in the cytoplasmic compartment, and 4.1R135+17b expression restores cell–cell contact localization of βII-spectrin. Bar, 10 μm. D and E, 4.1R+17b knockdown was achieved by exon 17b-specific siRNA (siRNA), and rescue was accomplished by co-expression rescue construct 4.1R135+17b (siRNA 4.1Rres). A scramble nontargeting siRNA served as a control (Ctrl). D, siRNA-mediated knockdown of 4.1R+17b forms reduces 4.1R135+17b and 4.1R80+17b expression, and rescue construct restores 4.1R135+17b expression in MDCK. Cell lysates from MDCK with indicated treatments were analyzed with an anti-ex17b antibody. GAPDH served as a loading control. Molecular mass markers (kDa) are provided. E, knockdown of 4.1R+17b forms delays the initial recruitment of E-cadherin to cell–cell contacts, and 4.1R135+17b expression restores the normal reassembly process. MDCK cells treated as indicated and subjected to calcium switch were immunofluorescently-stained for E-cadherin. Note: E-cadherin localizes at cell peripheries at early time points (0.5 and 1 h) in both control and rescue cells but not in ex17b–siRNA knockdown cells. E-cadherin recruits to cell border of all treatments 4 h after calcium repletion. Bar, 10 μm. F, quantification of percent of junction assembly after calcium switch. Data are shown as mean ± S.E. of 50 cells. Asterisks indicate statistical significance (***, p < 0.001).