Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis.
A stable 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.
Tavormina
Penny A
PA
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Côme
Marie-George
MG
Hudson
Joanna R
JR
Mo
Yin-Yuan
YY
Beck
William T
WT
Gorbsky
Gary J
GJ
D000818:Animals; D000951:Antigens, Neoplasm; D015153:Blotting, Western; D002460:Cell Line; D002875:Chromosomes / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D049452:Green Fluorescent Proteins; D018386:Kinetochores / Q000378:metabolism*; D007834:Lasers; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004250:DNA Topoisomerases, Type II