Nuclear scaffold proteins are differently sensitive to stabilizing treatment by heat or Cu++.
The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (M(r), 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125 and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg+2 ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold.
Neri
L M
LM
Istituto di Anatomia Umana Normale, Università di Ferrara, Italy.
Riederer
B M
BM
Marugg
R A
RA
Capitani
S
S
Martelli
A M
AM
D034961:Antigens, Nuclear; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D015415:Biomarkers / Q000737:chemistry; D015153:Blotting, Western; D003300:Copper / Q000494:pharmacology*; D006358:Hot Temperature; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D018613:Microscopy, Confocal; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D001324:Autoantigens; D015415:Biomarkers; D009687:Nuclear Proteins; D003300:Copper