Role of catenins in the development of gap junctions in rat cardiomyocytes.
Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of alpha-catenin, beta-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca(2+) (<5 microM) medium, then shifted to 1.8 mM Ca(2+) medium (Ca(2+) switch). Diffuse cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca(2+) medium. Staining of alpha-catenin, beta-catenin, and ZO-1 was detected at the plasma membrane of cell-cell contact sites 10 min after Ca(2+) switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca(2+) switch. Distinct junctional and extensive cytoplasmic staining of alpha-catenin, beta-catenin, ZO-1, and Cx43 was seen 2 h after Ca(2+) switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-beta-catenin antibodies showed that beta-catenin was associated with alpha-catenin, ZO-1, and Cx43 at 2 h after Ca(2+) switch. Intracellular application of antisera against alpha-catenin, beta-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca(2+) medium inhibited the redistribution of Cx43 to the plasma membrane following Ca(2+) switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.
Wu
Jiahn-Chun
JC
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan. jcwu@ha.mc.ntu.edu.tw
Tsai
Ru-Yin
RY
Chung
Tun-Hui
TH
D000818:Animals; D002118:Calcium; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018031:Connexin 43 / Q000032:analysis / Q000096:biosynthesis; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D018274:Electroporation; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D010750:Phosphoproteins / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D015534:Trans-Activators / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein; D051178:alpha Catenin; D051176:beta Catenin
D002413:Cations, Divalent; D018031:Connexin 43; C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C569044:Tjp1 protein, rat; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051178:alpha Catenin; D051176:beta Catenin; D002118:Calcium