Redistribution of DNA topoisomerase II β after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase IIβ (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37°or 42°C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0°C in a buffer containing spermine/spermidine/KC1 or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0°C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37°or 42°C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KC1, the incubation buffer also contained 5 mM Mg++ and the temperature was 37°C. If the stabilization was performed at 42°C, Mg++ failed to maintain the original distribution of DNA topoisomerase IIβ, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37°C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37°C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0°C incubated nuclei; they showed a certain degree of shrinkage in 0°C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37°or 42°C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37°C, but not at 42°C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.