Subnuclear localization of S/MAR-binding proteins is differently affected by in vitro stabilization with heat or Cu2+.

Abstract	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 extraction steps. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions, but must rather be stabilized before the application of extracting agents such as high salt solutions or lithium diiodosalicylate. We have examined the effect of two widely used stabilization procedures on the localization of nuclear matrix proteins. Four individual polypeptides were studied, all of which are scaffold or matrix-associated region (S/MAR)-binding proteins: SATB1, SAF-A/hnRNP-U, NuMA , and topoisomerase II alpha. Nuclei were isolated from K562 human erythroleukemia cells in a buffer containing spermine, spermidine, KCl and EDTA, and the nuclear matrix or scaffold was obtained by extraction with lithium diiodosalicylate after stabilization by heat treatment (37 degrees or 42 degrees C) or incubation with Cu2+ ions. When the localization of individual proteins was determined 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 stabilization protocols employed for the preparation of the nuclear matrix is desirable, especially by performing morphological controls.
Authors	L M Neri, F O Fackelmayer, M Zweyer, T Kohwi-Shigematsu, A M Martelli
Journal	Chromosoma (Chromosoma) Vol. 106 Issue 2 Pg. 81-93 (Jul 1997) ISSN: 0009-5915 [Print] Austria
PMID	9215557 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	Antigens, Neoplasm Antigens, Nuclear Cations, Divalent Cell Cycle Proteins DNA-Binding Proteins Heterogeneous-Nuclear Ribonucleoprotein U Heterogeneous-Nuclear Ribonucleoproteins Isoenzymes Matrix Attachment Region Binding Proteins NUMA1 protein, human Nuclear Matrix-Associated Proteins Nuclear Proteins Ribonucleoproteins SATB1 protein, human Copper DNA Topoisomerases, Type II
Topics	Antigens, Neoplasm Antigens, Nuclear Artifacts Cations, Divalent (pharmacology) Cell Cycle Proteins Cell Nucleus (chemistry, metabolism, ultrastructure) Copper (pharmacology) DNA Topoisomerases, Type II (isolation & purification) DNA-Binding Proteins (isolation & purification) Heterogeneous-Nuclear Ribonucleoprotein U Heterogeneous-Nuclear Ribonucleoproteins Histocytological Preparation Techniques Hot Temperature Humans Isoenzymes (isolation & purification) Lasers Leukemia, Erythroblastic, Acute Matrix Attachment Region Binding Proteins Microscopy, Confocal Nuclear Matrix (metabolism, ultrastructure) Nuclear Matrix-Associated Proteins Nuclear Proteins (isolation & purification) Protein Binding Ribonucleoproteins (isolation & purification) Tumor Cells, Cultured

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