ASSEMBLY OF SARCOPLASMIC-RETICULUM - LOCALIZATION BY IMMUNOFLUORESCENCE OF SARCOPLASMIC-RETICULUM PROTEINS IN DIFFERENTIATING RAT SKELETAL-MUSCLE CELL-CULTURES

Immunofluorescent staining techniques were used to study the distribution of the Ca2+ + Mg2+-dependent ATPase and calsequestrin in primary cultures of differentiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture, whereas the ATPase was not detected until 60 h. After cell fusion began, both proteins were identified in all multinucleated cells. Myoblasts grown for longer than 60 h in low Ca2+ medium contained calsequestrin and the ATPase, even though they were unable to fuse. Immunofluorescent staining of myoblasts showed that calsequestrin first appeared in a well-defined region of the cell near 1 end of the nucleus. At later times, the staining occupied progressively larger regions adjacent to the nucleus and took on a fibrous appearance. Calsequestrin first accumulated in the Golgi region and then gradually spread throughout the cell. The ATPase appeared to be concentrated in many small patches or foci throughout the cytoplasm and was never confined to 1 particular region, although some parts of the cell often stained more intensely than others. In multinucleated cells, alternating dark and fluorescent strands parallel to the longitudinal axis of the cells were evident. Fluorescent staining with these antisera was not observed in fibroblasts which were also present in the cultures.