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doi: 10.1242/10.1242/jcs.00258

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The organization of adherens junctions and desmosomes at the cardiac intercalated disc is independent of gap junctions

Division of Cardiology, Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

View larger version (56K): [in a new window]   Fig. 1. Immunoblotting of intercalated disc-associated proteins in Cx43 CKO hearts. Immunoblots of total ventricular lysates using specific antibodies directed against components of the cardiac intercalated disc. Proteins detected are indicated to the left of each blot. Immunoblots represent either a re-probed or duplicate blot, with the same heart lysate loaded on the same lane for each of the different antibodies.

View larger version (56K): [in a new window]   Fig. 2. Subcellular localization of ß-catenin and p120 catenin in Cx43 CKO hearts. (A) Immunoblot analysis of cytosol fractions from control and CKO mouse hearts for the presence of ß-catenin and p120 catenin. (B) Immunoblot analysis of neat plasma membrane fractions from control and CKO mouse hearts for the presence of ß-catenin and p120 catenin. Coomassie staining was used to ensure equivalent loading of protein per lane in the cytosol and plasma membrane blots. (C) Immunoprecipitation of plasma membrane fractions with an anti-pan-cadherin antibody followed by blotting with an anti-p120 catenin antibody demonstrates an association of cadherin with p120 in control and CKO hearts.

View larger version (128K): [in a new window]   Fig. 3. Immunofluorescent staining for cadherin in control and Cx43 CKO mouse heart sections. Heart sections were double stained with a mouse anti-pan-cadherin antibody (A, control; D, CKO), and a rabbit anti-Cx43 antibody (B, control; E, CKO) and imaged with a confocal microscope. Merged images are shown in C (control) and F (CKO) and demonstrate that adherens junction localization to the intercalated disc, indicated by cadherin staining, is unchanged in the absence of Cx43. Magnification, 40x; bar, 50 µm.

View larger version (114K): [in a new window]   Fig. 4. Immunofluorescent staining for desmoplakin in control and Cx43 CKO mouse heart sections. Heart sections are double stained for desmoplakin (A, control; D, CKO) and Cx43 (B, control; E, CKO) and imaged with a confocal microscope. Merged images are shown in C (control) and F (CKO). No change in the pattern of desmoplakin staining in CKO hearts is evident in comparison with controls. Magnification, 40x; bar, 50 µm.

View larger version (108K): [in a new window]   Fig. 5. Immunofluorescent images of desmoplakin and cadherin double-stained control and CKO hearts. Shown are stacked confocal images through the intercalated discs in en-face orientation (arrows) in mouse heart sections double stained for desmoplakin (A, control; D, CKO) and cadherin (B, control; E, CKO). Merged images (C, control; F, CKO) suggest that desmoplakin and cadherin are juxtaposed, but in general not co-localized, at the intercalated discs, in a similar pattern in both control and CKO hearts. Magnification, 63x; zoom, 2.96; bar, 10 µm.

View larger version (119K): [in a new window]   Fig. 6. p120 co-localizes with cadherin at the intercalated disc in control and CKO hearts. Stacked confocal images of heart sections double stained for p120 catenin (A, control; D, CKO) and cadherin (B, control; E, CKO) show co-localization (arrows) at the intercalated disc (merged images in C, control; F, CKO). Magnification, 63x; zoom, 2.96; bar, 10 µm.

View larger version (138K): [in a new window]   Fig. 7. Co-localization of ß-catenin with cadherin and plakoglobin with desmoplakin at the intercalated disc in control and CKO mouse hearts. Frozen sections were double stained for cadherin (A, control; D, CKO) and ß-catenin (B, control; E, CKO) or desmoplakin (G, control; J, CKO) and plakoglobin (H, control; K, CKO). Merged images of ß-catenin and cadherin double staining are shown in C (control) and F (CKO) and those of plakoglobin and desmoplakin in I (control) and L (CKO). All images represent stacked confocal slices taken every 0.5 µm through the tissue section at a magnification of 63x and a zoom factor of 1.00. Bar, 30 µm.

View larger version (203K): [in a new window]   Fig. 8. Immunofluorescent staining for ZO-1 and vinculin in control and CKO mouse heart sections. ZO-1 (A, control; B, CKO) and vinculin (C, control; D, CKO) staining revealed similar patterns in control and CKO hearts. Magnification, 40x; bar, 50 µm.

View larger version (204K): [in a new window]   Fig. 9. Electron microscopy of the intercalated disc in control and CKO mice. Electron microscopy of the intercalated disc in a control mouse heart (A) shows a gap junction (arrow) in close proximity to adherens junctions (white arrowheads) and desmosomes (black arrowheads), while a representative intercalated disc in a CKO heart (B) shows no gap junctions but intact architecture of the remaining structures. Immuno-electron microscopy for Cx43 in a control heart (C) confirms the presence of Cx43 in the cardiac gap junction and demonstrates the absence of Cx43 label at the intercalated disc of a CKO mouse (D). Immuno-electron microscopy for cadherin in controls (E) and CKO hearts (F) demonstrates intact adherens junctions in both samples. Bars, 50 nm (A,B); 140 nm (C); 70 nm (D); 95 nm (E); 70 nm (F).