Figure 1.

GT2-LPk cells express EGFP-topoIIα. (A) Immunoblotting with anti-topoIIα antibody of whole-cell extracts of parental LLC-Pk cells and GT2-LPk. GT2-LPk cells show downregulated expression of endogenous topoIIα. (B) EGFP–topoIIα immunoprecipitated from GT2-LPk cells catalyzes decatenation of kinetoplast DNA. Lanes contain respectively decatenated control, kinetoplast DNA with no topoisomerase II added, and two separate immunoprecipitations of GT2-LPk extracts with anti-GFP antibody. Arrows indicate the migration of the decatenated products (nicked and supercoiled). (C) EGFP– topoIIα localization in a panel of living cells at various stages of mitosis. EGFP–topoIIα associates with chromosomes in prophase, becomes concentrated at kinetochores (closed arrows) and along chromosome arms in prometaphase and metaphase, and associates with chromosome arms in anaphase. In late anaphase and telophase, EGFP–topoIIα increases in the cytoplasm with some becoming concentrated in cytoplasmic foci (open arrow). (D) Imaging of fixed GT2-LPk cell at late prometaphase after staining with DAPI shows localization of topoisomerase II to the kinetochore region and axes of chromosomes. Cells were simultaneously fixed and permeabilized by treatment with 2% formaldehyde, 0.5% TX-100 in microtubule stabilizing buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO₄, pH 6.95). The images represent a maximum projection of six 0.2-μm optical sections. In the merged image, EGFP–topoIIα is green and appears yellow due to overlap with the DNA in red. Bars, 10 μm.