Mandinova A, Atar D, Schäfer B W, Spiess M, Aebi U, Heizmann C W
Maurice E. Müller-Institute, Biocentrum, University of Basel, Switzerland.
J Cell Sci. 1998 Jul 30;111 ( Pt 14):2043-54. doi: 10.1242/jcs.111.14.2043.
Changes in cytosolic Ca2+ concentration control a wide range of cellular responses, and intracellular Ca2+-binding proteins are the key molecules to transduce Ca2+ signaling via interactions with different types of target proteins. Among these, S100 Ca2+-binding proteins, characterized by a common structural motif, the EF-hand, have recently attracted major interest due to their cell- and tissue-specific expression pattern and involvement in various pathological processes. The aim of our study was to identify the subcellular localization of S100 proteins in vascular smooth muscle cell lines derived from human aorta and intestinal smooth muscles, and in primary cell cultures derived from arterial smooth muscle tissue under normal conditions and after stimulation of the intracellular Ca2+ concentration. Confocal laser scanning microscopy was used with a specially designed colocalization software. Distinct intracellular localization of S100 proteins was observed: S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found predominantly in the cytosol where they were strongly associated with the sarcoplasmic reticulum and with actin stress fibers. In contrast, S100A2 was located primarily in the cell nucleus. Using a sedimentation assay and subsequent electron microscopy after negative staining, we demonstrated that S100A1 directly interacts with filamentous actin in a Ca2+-dependent manner. After thapsigargin (1 microM) induced increase of the intracellular Ca2+ concentration, specific vesicular structures in the sarcoplasmic reticulum region of the cell were formed with high S100 protein content. In conclusion, we demonstrated a distinct subcellular localization pattern of S100 proteins and their interaction with actin filaments and the sarcoplasmic reticulum in human smooth muscle cells. The specific translocation of S100 proteins after intracellular Ca2+ increase supports the hypothesis that S100 proteins exert several important functions in the regulation of Ca2+ homeostasis in smooth muscle cells.
胞质Ca2+浓度的变化控制着广泛的细胞反应，细胞内Ca2+结合蛋白是通过与不同类型靶蛋白相互作用来转导Ca2+信号的关键分子。其中，以共同结构基序EF手为特征的S100 Ca2+结合蛋白，由于其细胞和组织特异性表达模式以及参与各种病理过程，最近引起了人们的极大兴趣。我们研究的目的是确定S100蛋白在源自人主动脉和肠道平滑肌的血管平滑肌细胞系以及在正常条件下和细胞内Ca2+浓度刺激后源自动脉平滑肌组织的原代细胞培养物中的亚细胞定位。使用共聚焦激光扫描显微镜和专门设计的共定位软件。观察到S100蛋白有明显的细胞内定位：S100A6存在于肌浆网以及细胞核中。S100A1和S100A4主要存在于胞质溶胶中，它们与肌浆网和肌动蛋白应力纤维密切相关。相比之下，S100A2主要位于细胞核中。通过沉降试验以及阴性染色后的后续电子显微镜观察，我们证明S100A1以Ca2+依赖的方式直接与丝状肌动蛋白相互作用。在毒胡萝卜素（1 microM）诱导细胞内Ca2+浓度升高后，细胞肌浆网区域形成了具有高S100蛋白含量的特定囊泡结构。总之，我们证明了S100蛋白在人平滑肌细胞中有明显的亚细胞定位模式，以及它们与肌动蛋白丝和肌浆网的相互作用。细胞内Ca2+升高后S100蛋白的特异性转位支持了S100蛋白在平滑肌细胞Ca2+稳态调节中发挥多种重要功能的假说。
