Ioshii S O, Imanaka-Yoshida K, Yoshida T
Department of Pathology, Mie University School of Medicine, Japan.
J Cell Physiol. 1994 Jan;158(1):87-96. doi: 10.1002/jcp.1041580112.
The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca2+ ions in muscle cells. Calsequestrin is a major Ca(2+)-storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin-positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine-phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross-striations at Z-lines. When newborn cardiomyocytes were isolated, calsequestrin-positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin-positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross-striations at the Z-lines. Double-labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin-positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin-positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR.
肌浆网（SR）调节肌肉细胞中细胞质游离Ca2+离子的水平。肌集钙蛋白是一种主要的Ca(2+)储存蛋白，定位于肌浆网的特殊部位。为了研究肌集钙蛋白阳性肌浆网的发育及其与细胞骨架的相互作用，我们用抗肌集钙蛋白和抗微管蛋白抗体以及罗丹明 - 鬼笔环肽通过免疫荧光法检测了新生大鼠培养心肌细胞中肌集钙蛋白的分布。在新生大鼠心脏的冰冻切片中，抗肌集钙蛋白免疫染色在Z线处表现为横纹。当分离新生心肌细胞时，肌集钙蛋白阳性的肌浆网紊乱，在肌膜下表现为小泡，而肌原纤维聚集在细胞中央。随着细胞在培养中铺展，肌集钙蛋白阳性小泡扩散到细胞质周边，与发育中的肌原纤维相关联。在成熟细胞中，肌集钙蛋白与肌原纤维紧密相关，在Z线处呈现横纹。使用抗肌集钙蛋白和抗微管蛋白抗体进行双重标记表明，肌集钙蛋白阳性结构的分布与微管阵列的分布相似。当在早期用诺考达唑使微管解聚时，肌浆网向细胞周边的延伸受到抑制。在成熟心肌细胞中，诺考达唑似乎不影响肌浆网的分布。这些结果表明，心肌细胞中肌集钙蛋白阳性的肌浆网在肌原纤维形成过程中在肌原纤维的适当部位组装，并且微管可能作为肌浆网成分运输的通道。
