Schafer D A, Waddle J A, Cooper J A
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri.
Cell Motil Cytoskeleton. 1993;25(4):317-35. doi: 10.1002/cm.970250403.
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.
在肌原纤维形成过程中，肌动蛋白丝的组织结构会发生显著变化。在成熟的骨骼肌中，CapZ和肌动蛋白丝的尖端均位于Z盘。在体外，CapZ结合肌动蛋白丝的尖端，阻止肌动蛋白亚基的添加和丢失；CapZ还能在体外引发肌动蛋白聚合。综合这些特性表明，CapZ可能在肌原纤维形成过程中发挥组织肌动蛋白丝的作用。我们在此报告，成年鸡胸肌肌原纤维中CapZ的量足以“帽化”肌节中的每一根肌动蛋白丝。利用培养的骨骼肌细胞的双重免疫荧光显微镜技术，确定了CapZ在肌原纤维形成过程中相对于肌动蛋白、α - 辅肌动蛋白、肌联蛋白和肌球蛋白的空间和时间分布。特别令人感兴趣的是CapZ在新生Z盘处的组装与新生肌原纤维中肌动蛋白丝组织的关系。在成肌细胞和年轻的肌管中，CapZ在细胞质中呈弥散分布。随着肌管成熟，最初观察到CapZ沿称为应力纤维样结构（SFLS）的无条纹肌动蛋白丝呈均匀分布。在肌动蛋白出现条纹状染色模式之前，CapZ沿SFLS以成熟Z盘特有的周期性模式被观察到。在较老的肌管中，当观察到肌动蛋白呈I带特有的模式时，CapZ以成熟Z盘特有的周期性模式分布。CapZ在肌动蛋白出现条纹状模式之前就在新生Z盘处组装这一发现，与CapZ在骨骼肌细胞I带形成过程中指导肌动蛋白丝的位置和极性的假设一致。相对于肌节α - 辅肌动蛋白、肌联蛋白和粗肌丝的组装，也观察到CapZ在新生Z盘结构处的组装。在CapZ出现在新生Z盘之前，在具有成熟肌节组织的结构中观察到了肌联蛋白和肌球蛋白。在年轻肌管中，CapZ和肌节α - 辅肌动蛋白的分布不一致；在较老的肌管中，CapZ和α - 辅肌动蛋白都共定位于Z盘。在心肌细胞中，CapZ在Z盘处被检测到，并在整个细胞质中呈点状分布。CapZ还与A - CAM和纽蛋白在心肌细胞形成的细胞 - 细胞连接处共定位。
