SUBNUCLEAR LOCALIZATION OF S MAR-BINDING PROTEINS IS DIFFERENTLY AFFECTED BY IN-VITRO STABILIZATION WITH HEAT OR CU2+/

The nuclear matrix, a proteinaceous entity thought to be a scaffolding structure that determines the higher order organization of eukaryotic chromatin, is usually prepared from intact nuclei by a series of extr action steps. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions, but must rather be stabil ized before the application of extracting agents such as high salt sol utions or lithium diiodosalicylate. We have examined the effect of two widely used stabilization procedures on the localization of nuclear m atrix proteins. Four individual polypeptides were studied, all of whic h are scaffold or matrix-associated region (S/MAR)-binding proteins: S ATB1, SAF-A/hnRNP-U, NuMA, and topoisomerase II alpha. Nuclei were iso lated from K562 human erythroleukemia cells in a buffer containing spe rmine, spermidine, KCl and EDTA, and the nuclear matrix or scaffold wa s obtained by extraction with lithium diiodosalicylate after stabiliza tion by heat treatment (37 degrees or 42 degrees C) or incubation with Cu2+ ions. When the localization of individual proteins was determine d by immunofluorescent staining and confocal scanning laser microscopy , markedly different consequences of the two stabilization strategies became evident, ranging from a total maintenance of the localization ( NuMA and topoisomerase II alpha) to a marked redistribution (SATB1 and SAF-A/hnRNP-U). Our results seem to indicate that a reevaluation of s tabilization protocols employed for the preparation of the nuclear mat rix is desirable, especially by performing morphological controls.