Circular nuclear alignment in multinucleate PC12D cells produced by cell fusion with polyethylene glycol

PC12D cells, a subline of PC12 cells, extend neurites within a few hours in response to nerve growth factor (NGF). Multinucleate PC12D cells were produced by cell fusion with polyethylene glycol (PEG). The syncytia extended large neurites following treatment with NGF. In the syncytia, nuclei were distributed in a circular alignment. Many nuclear rings were found in the culture dish 1 day after treatment with PEG, although they were sometimes seen in syncytia within several hours. Colchicine prevented this alignment or disrupted the clusters when added subsequent to their formation. Cytochalasin B did not prevent the formation of these rings, nor affect the alignment of nuclei. Immnofluorescent staining of γ-tubulin, an integral protein of the centrosome, revealed intense dot-like structures in the center of nuclear rings in syncytia. Twice as many dots, or centrioles, as nuclei were present in each syncytium. However, most microtubules were localized around the nuclei and did not appear to extend from the cluster of centrioles. Fluorescent labeling of mitochondria showed them to mainly be localized inside the nuclear rings. Meanwhile, immunofluorescently stained endoplasmic reticulum was mainly localized along with the circular alignment of nuclei. Therefore, not only nuclei but various cell organelles are situated in each characteristic location in the syncytia. These data suggest that microtubules are involved in the migration and alignment of nuclei and presumably of cell organelles in the syncytia.