Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix

The higher order of chromatin organization is thought to be determined by t he nuclear matrix, a mainly proteinaceous structure that would act as a nuc leoskeleton. The matrix is obtained from isolated nuclei by a series of ext raction steps involving the use of high salt and nonspecific nucleases, whi ch remove chromatin and other loosely bound components. It is currently und er debate whether these structures, isolated in vitro by unphysiological ex traction buffers. correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist t hese extractions steps; rather, it must be stabilized before the applicatio n of extracting agents. In this study nuclei, isolated from K562 human eryt hroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, w e determined that except for Ca2+, all the other metals induced a stabiliza tion of the matrix, which retained the inner fibrogranular network and resi dual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide patter n, characteristic of each type of stabilizing ion employed. We also investi gated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisom erase II alpha, and RNP. Confocal microscopy analysis showed that only NuMa , and, to a lesser extent, topoisomerase II alpha, were unaffected by stabi lization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate thai several metal ion s are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoiso merase II alpha may act as structural components of the nuclear matrix. J. Cell. Biochem. 73:342-354, 1999. (C) 1999 Wiley-Liss, Inc.