Localization of CapZ during myofibrillogenesis in cultured chicken muscle

Abstract

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, a-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 2-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 a-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 a-actinin in young myotubes was not coincident; in older myotubes, both CapZ and a-actinin were co-localized at 2-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 rnyocytes. 0 1993 Wiley-Liss, Inc. Address reprint requests to Dr. Dorothy A. Schafer, Department of versity School of Medicine, 660 s. Euclid A~~~~ St, Louis, MO known about molecular Of this process* Cell Biology and physiology, Campus Box 8228, Washington Uniaments changes from non-striated bundles of longitudi-0 1993 Wiley-Liss, Inc. 631 lo.

Models of Thin Filament Assembly in Cardiac and Skeletal Muscle.

“…Since all of the barbed ends of the thin filaments are precisely aligned in mature Z-disks, the variability in length seen in cardiac muscle must result from differential regulation at the pointed ends of the filaments (in the center region of the sarcomere) (24). Consistent with the strict alignment of the barbed ends of the filaments in both cardiac and skeletal muscle, capZ assembles early during myofibril assembly in both primary cultures of chick embryonic skeletal myogenic cells and cardiac myocytes [that is, before the appearance of actin filament striations (25,26)]. This observation, together with the characteristics of capZ deduced from in vitro studies (2, 4), suggests that capZ functions to nucleate actin filament polymerization at the Z-line, and to organize (e.g., establish polarity) and maintain thin filament length at the barbed end from the earliest stages of myofibril assembly in both of these striated muscle cell types.…”

Differential effects of latrunculin-A on myofibrils in cultures of skeletal muscle cells: Insights into mechanisms of myofibrillogenesis

“…The capping protein CapZ binds the pointed or fast-growing end of actin filaments embedded in the Zbands of mature myofibrils [Casella et al, 1987;Schafer et al, 1993]. Myotubes were stained with an antibody directed against CapZ, in order to determine at which of the three different stages of myofibrillogenesis this actinstabilizing protein first appeared.…”

Actin Capping Protein

“…CP-␤ 1 assembles at Z-lines before the formation of actin filaments in developing myotubes. 4 Disruption of actin-CP-␤ 1 interaction impairs myofibrillogenesis 5 and produces gross myofibrillar disarray. 2 Although the importance of CP-␤ 1 in the development of normal muscle architecture is well established, its role in muscle function has not been investigated.…”