DISTRIBUTION OF A CA2- AN IMMUNOCYTOCHEMICAL STUDY USING AN ANTIBODY AGAINST CALRETICULIN( STORING SITE IN PTK2 CELLS DURING INTERPHASE ANDMITOSIS )

To study the distribution of a major CA(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase a nd mitosis, we prepared an affinity-purified polyclonal antibody again st bovine liver calreticulin (CRT), a major Ca2+-binding protein of th e endoplasmic reticulum (ER). Immunofluorescence microscopy and immuno peroxidase electron microscopy showed that the anti-CRT antibody label ed a continuous reticular network of the ER and the nuclear envelope i n interphase PtK2 cells. The same PtK2 cells double-stained with DiOC( 6) (3) and the anti-CRT antibody revealed labeling of identical reticu lar membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These resu lts confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in nonmuscle cells. In mito tic cells, marked changes of the labeled structure began at prophase-p rometaphase and persisted throughout all phases of mitosis. The cytopl asm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microsco py 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 b e responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during int erphase.