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. 2019 Nov 27;295(1):191–211. doi: 10.1074/jbc.RA119.009650

Figure 7.

Figure 7.

Expression of 4.1R+17b or 4.1R−17b forms does not impede the protein levels of AJ and spectrin–actin cytoskeleton, but 4.1R−17b expression affects the organization of the spectrin lattice in the area of cell–cell contacts. MDCK cells transfected with EGFP–4.1R+17b or EGFP–4.1R−17b forms were analyzed for AJ and spectrin–actin protein levels as well as for the intracellular localization of actin and βII-spectrin 36 h post-transfection. A, Western blotting analyses for AJ and actin skeleton proteins from MDCK cells transfected with EGFP–4.1R80, EGFP–4.1R80+17b, EGFP–4.1R135, or EGFP–4.1R135+17b. Equal amounts of lysates from each transfected culture were immunoblotted with anti-GFP, α-spectrin, βII-spectrin, β-actin, E-cadherin, or β-catenin Ab. GAPDH served as a loading control. Molecular mass markers (kDa) are provided. B, F-actin and βII-spectrin co-localize with β-catenin at the AJs of MDCK cells. MDCK were plated on coverslips and stained with Alexa Fluor 488 phalloidin for actin or anti-βII-spectrin Ab for βII-spectrin. Both were dual-immunolabeled for β-catenin and revealed with a Zeiss microscope. Bar, 10 μm. C, reduction of actin at AJs of EGFP–4.1R135–expressing cells but not that of EGFP–4.1R135+17b–expressing cells. MDCK cells transfected with EGFP–4.1R135+17b or EGFP–4.1R135 were labeled with Alexa Fluor 568 phalloidin for actin. D, EGFP–4.1R135 but not EGFP–4.1R135+17b expression transforms junctional localization of βII-spectrin into diffuse cytoplasmic accumulation. MDCK cells transfected with EGFP–4.1R135+17b or EGFP–4.1R135 were labeled for the presence of βII-spectrin. Bar, 10 μm. E, presence of actin at the cell–cell contacts coincides with that of E-cadherin during the AJ reassembly process. MDCK cells were stained for actin and E-cadherin at the indicated time after calcium repletion. Note: fluorescence labeling shows formation of the circumferential F-actin and E-cadherin at the AJs after 0.5 h of calcium repletion (arrows) and persists through the AJ maturation process. Bar, 10 μm. F, ratio of the fluorescence intensity of E-cadherin and actin at the cell–cell junction (red boxed region of panel E) versus adjacent cytoplasm (yellow boxed region of panel E) was measured (n = 50). (F.I., fluorescence intensity; Mem, membrane; Cyto, cytoplasm). Values are mean ± S.E.