Koyabu S, Imanaka-Yoshida K, Ioshii S O, Nakano T, Yoshida T
First Department of Internal Medicine, Mie University School of Medicine, Japan.
Cell Motil Cytoskeleton. 1994;29(3):259-70. doi: 10.1002/cm.970290309.
A major Ca(2+)-storing protein in endoplasmic reticulum (ER) of non-muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca(2+)-binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti-calsequestrin, anti-calreticulin, anti-desmin, and anti-sarcomeric myosin antibodies and rhodamine-phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin-positive cells demonstrated the reticular staining of calreticulin, as did desmin-negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin-positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti-calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation.
非肌肉细胞内质网（ER）中一种主要的钙储存蛋白是钙网蛋白（CR），它被认为在功能上与肌集钙蛋白同源。肌集钙蛋白是横纹肌肌浆网（SR）中的一种钙结合蛋白，在肌肉舒张时储存钙离子。为了研究肌集钙蛋白和钙网蛋白在骨骼肌分化过程中的表达和分布，使用抗肌集钙蛋白、抗钙网蛋白、抗结蛋白和抗肌节肌球蛋白抗体以及罗丹明-鬼笔环肽，通过免疫荧光观察培养的鸡胚骨骼肌。培养6小时内，成肌细胞开始表达结蛋白。结蛋白阳性细胞和结蛋白阴性细胞一样，都显示出钙网蛋白的网状染色。在融合前后，肌集钙蛋白和肌节肌球蛋白开始出现在结蛋白阳性细胞中。肌集钙蛋白的表达略早于肌节肌球蛋白。随着肌管成熟，肌集钙蛋白的荧光点增多，并沿肌原纤维扩散到细胞周边，而钙网蛋白的网状模式逐渐消失。双重标记显示肌集钙蛋白与钙网蛋白共定位。在成熟的肌管中，抗肌集钙蛋白染色显示沿肌原纤维有许多点，而除了核周区域外几乎看不到钙网蛋白。这些结果表明，在骨骼肌分化过程中，肌集钙蛋白和钙网蛋白的表达发生了转换。
