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Published 8 July 2002. doi:10.1083/jcb.200202053
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© The Rockefeller University Press, 0021-9525/2002/7/23 $5.00
The Journal of Cell Biology, Volume 158, Number 1, July 8, 2002 23-29

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 Rapid exchange of mammalian topoisomerase II{alpha} at kinetochores and chromosome arms in mitosis

Penny A. Tavormina1,2, Marie-George Côme1, Joanna R. Hudson1, Yin-Yuan Mo3, William T. Beck3 and Gary J. Gorbsky1

1 Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104
2 Universal Imaging Corporation, Downingtown, PA 19335
3 Department of Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, Chicago, IL 60612

Address correspondence to Gary J. Gorbsky, Dept. of Cell Biology, University of Oklahoma Health Sciences Center, Biomedical Research Center, Rm. 266, 975 NE 10th St., Oklahoma City, OK 73104. Tel.: (405) 271-3486. Fax: (405) 271-7158. E-mail: GJG{at}ouhsc.edu

Astable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase II{alpha} (topoII{alpha}) protein was downregulated and replaced by the expression of topoII{alpha} fused with enhanced green fluorescent protein (EGFP–topoII{alpha}). The EGFP–topoII{alpha} faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP–topoII{alpha} accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP–topoII{alpha} diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP–topoII{alpha} associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoII{alpha} was essential for rapid dynamics, as ICRF-187, an inhibitor of topoII{alpha}, blocked recovery after photobleaching. Although some topoII{alpha} may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoII{alpha} in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation.

Key Words: DNA topoisomerase II; cell division; cell cycle proteins; kinetochore; nucleolus


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