Ioshii S O, Yoshida T, Imanaka-Yoshida K, Izutsu K
Department of Pathology, Mie University School of Medicine, Japan.
Eur J Cell Biol. 1995 Jan;66(1):82-93.
To study the distribution of a major Ca(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase and mitosis, we prepared an affinity-purified polyclonal antibody against bovine liver calreticulin (CRT), a major Ca(2+)-binding protein of the endoplasmic reticulum (ER). Immunofluorescence microscopy and immunoperoxidase electron microscopy showed that the anti-CRT antibody labeled a continuous reticular network of the ER and the nuclear envelope in interphase PtK2 cells. The same PtK2 cells double-stained with DiOC6 (3) and the anti-CRT antibody revealed labeling of identical reticular membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These results confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in non-muscle cells. In mitotic cells, marked changes of the labeled structure began at prophase-prometaphase and persisted throughout all phases of mitosis. The cytoplasm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microscopy and immunoelectron microscopy demonstrated that the CRT-containing membranes changed to segmented tubuloreticular structures, which were concentrated around the mitotic spindle. The ER containing CRT could be responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during interphase.
为研究大鼠袋鼠肾上皮细胞系PtK2细胞在间期和有丝分裂期间主要Ca(2+)螯合位点的分布，我们制备了一种针对牛肝钙网蛋白（CRT）的亲和纯化多克隆抗体，钙网蛋白是内质网（ER）的一种主要Ca(2+)结合蛋白。免疫荧光显微镜和免疫过氧化物酶电子显微镜显示，抗CRT抗体标记了间期PtK2细胞中内质网和核膜的连续网状网络。用DiOC6(3)和抗CRT抗体对相同的PtK2细胞进行双重染色，显示相同网状膜的标记。与在内质网中的定位不同，线粒体和高尔基体未被标记。这些结果证实了钙网蛋白在内质网中的唯一定位，并且该细胞器是非肌肉细胞中Ca2+储存的主要位点。在有丝分裂细胞中，标记结构的显著变化始于前期-前中期，并在有丝分裂的所有阶段持续存在。有丝分裂细胞的细胞质显示弥漫性荧光，在有丝分裂纺锤体周围更强烈，但不在纺锤体内。共聚焦显微镜和免疫电子显微镜表明，含有钙网蛋白的膜转变为分段的管状网状结构，这些结构集中在有丝分裂纺锤体周围。含有钙网蛋白的内质网可能负责在有丝分裂期间以及间期螯合Ca2+并调节该阳离子的浓度。
