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First published online 25 February 2003
doi: 10.1242/jcs.00339

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Cadherin-mediated adhesion is essential for myofibril continuity across the plasma membrane but not for assembly of the contractile apparatus

Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

View larger version (149K): [in a new window]   Fig. 1. In vitro culture of myocytes derived from N-cadherin-null and E-cadherin-rescued N-cadherin-null embryos. Wild-type myocytes formed tightly adherent aggregates (A) after 3 days in culture. In comparison, small, less-compacted aggregates of N-cadherin-deficient myocytes were observed (B). Although the cells were less adhesive, small aggregates of mutant myocytes were observed beating synchronously, albeit more weakly compared with wild-type aggregates. Many mutant cells had difficulty remaining attached to the substrate, causing them to round up (C). Introduction of the MHC/Ecad transgene into the mutant background restored normal cell adhesion, resulting in large aggregates of strongly beating myocytes (D). Bar, 10 µm.

View larger version (101K): [in a new window]   Fig. 2. Expression of ß-catenin and p120ctn in transgenic myocytes. Myocyte cultures were examined by indirect immunofluorescence for expression of ß-catenin (A-C) and p120ctn (D-F). Both catenins showed localization at cell-cell boundaries in wild-type cells (A,D). Expression of ß-catenin (B) and p120ctn (E) were significantly reduced in the N-cadherin-null myocytes. Both catenins (C,F) were found localized to cell-cell contacts in the mutant myocytes expressing the MHC/Ecad transgene. Bar, 50 µm.

View larger version (152K): [in a new window]   Fig. 3. Myofibrillogenesis in N-cadherin-null myocytes. Wild-type (A,B) and mutant (C,D) myocytes were double stained for F-actin (A,C) and sarcomeric -actinin (B,D). Overall, myofibril structure appeared remarkably similar in these well-spread myocytes, as illustrated by the striated pattern of the myofibril components. Based on the phalloidin stain, it did appear that the mutant myofibrils were somewhat thicker or denser compared with the wild-type structures; however, no apparent difference was observed with -actinin staining. Bar, 50 µm.

View larger version (117K): [in a new window]   Fig. 6. Focal contact distribution in transgenic myocytes. Integrin-mediated cell-substrate adhesion is responsible for proper cell spreading and motility. Focal adhesions were visualized in wild-type (A,B), N-cadherin-null (C,D) and rescued (E,F) myocytes using antibodies against ß1 integrin (A,C,E) and vinculin (B,D,F). Although the N-cadherin-deficient cells often appeared less well spread compared with wild-type, the distribution of integrin and vinculin was similar between the mutant (C,D) and wild-type (A,B) cells. Focal contacts in mutant cells expressing E-cadherin also appeared normal (E,F). Bar, 50 µm.

View larger version (111K): [in a new window]   Fig. 4. Cadherin-mediated adhesion and myofibril alignment. Aggregates of wild-type (A-C), mutant (D-F) and rescued (G-L) myocytes were double stained for F-actin and either N-cadherin (A), desmosomal protein (D) or E-cadherin (G,J). In wild-type cells, myofibrils run in a linear fashion between neighboring myocytes with colocalization of their ends at regions of cell-cell contact as seen in the merged image (C). Myofibril disorganization is shown for mutant aggregates (E,F) from two independent experiments. In both cultures, the orientation of the myofibrils was random with respect to their neighboring cells (i.e. no continuity across the plasma membrane). E-cadherin-mediated cell adhesion restored proper alignment of the myofibrils between the N-cadherin-deficient myocytes (G-I) and E-cadherin localized to costameric structures on the dorsal surface of the myocyte (J). Bar, 50 µm.

View larger version (48K): [in a new window]   Fig. 5. Expression of Cx43 in transgenic myocytes. The presence of Cx43-containing gap junctions was determined by immunostaining wild-type (A), N-cadherin-null (B) and rescued (C) myocytes with a Cx43 antibody. A typical punctate pattern of Cx43 was observed in regions of cell-cell contact in the wild-type myocytes (A). Cx43 expression was reduced in N-cadherin-deficient myocytes (arrow, B). E-cadherin-mediated cell adhesion restored Cx43 expression to normal levels in the mutant myocytes (C). Bar, 50 µm.