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 i n intact K562 human erythroleukemia cells, isolated nuclei, and nuclea r scaffolds. Nuclear scaffolds were obtained by extraction with the io nic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of diva lent cations (metal-depleted nuclei) and stabilize d either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ io ns at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffe cted 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, unl ess stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other a ntigens (M(r) 125 and 126 kD), whereas in heat-stabilized nuclei we de tected dramatic changes. In nuclear scaffolds reacted with antibodies to 125- and 126-kD proteins, the fluorescent pattern was always disarr anged regardless of the stabilization procedure. These results, obtain ed with nuclei prepared in the absence of Mg++ ions, indicate that hea t treatment per se can induce changes in the distribution of nuclear p roteins, at Variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly beca use of its specific association with the nuclear scaffold.