Localization of CapZ during myofibrillogenesis in cultured chicken muscle.
Actin filaments undergo dramatic changes in their organization during myofibrillogenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z-discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to "cap" each actin filament of the sacromere. Double immunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, alpha-actinin, titin, and myosin during myofibrillogenesis. Of particular interest was the assembly of CapZ at nascent Z-discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non-striated actin filaments called stress fiber-like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z-discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I-bands, CapZ was distributed in a periodic pattern characteristic of mature Z-discs. The finding that CapZ was assembled at nascent Z-discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I-band formation in skeletal muscle cells. The assembly of CapZ at nascent Z-disc structures also was observed relative to the assembly of sarcomeric alpha-actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z-discs. The distribution of CapZ and sarcomeric alpha-actinin in young myotubes was not coincident; in older myotubes, both CapZ and alpha-actinin were co-localized at Z-discs. In cardiac myocytes, CapZ was detected at Z-discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co-localized with A-CAM and vinculin at cell-cell junctions formed by the myocytes.
Schafer
D A
DA
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri.
Waddle
J A
JA
Cooper
J A
JA
D000185:Actinin / Q000032:analysis / Q000276:immunology; D000199:Actins / Q000032:analysis / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D000918:Antibody Specificity; D051347:CapZ Actin Capping Protein; D002478:Cells, Cultured; D002642:Chick Embryo; D064211:Connectin; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000737:chemistry / Q000648:ultrastructure; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000032:analysis* / Q000276:immunology; D009132:Muscles / Q000737:chemistry* / Q000166:cytology / Q000196:embryology*; D009206:Myocardium / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D009210:Myofibrils / Q000737:chemistry / Q000648:ultrastructure; D009218:Myosins / Q000032:analysis / Q000276:immunology; D011494:Protein Kinases
D000199:Actins; D000906:Antibodies; D000911:Antibodies, Monoclonal; D051347:CapZ Actin Capping Protein; D064211:Connectin; D008840:Microfilament Proteins; D009124:Muscle Proteins; D000185:Actinin; D011494:Protein Kinases; D009218:Myosins