Structure and function studies of the nucleus
Czubryt, Michael Paul
Modern studies of the cell nucleus have provided a highly detailed view of its roles and the structures and mechanisms that carry out these roles, but many facets of nuclear physiology remain unclear. The present thesis focuses on three main areas of nuclear research. 'First', several calcium binding proteins in pig cardiac and rat liver nuclei were isolated and identified using high salt extraction followed by Stains-All staining and 45Ca2+ overlays of western blots. These proteins, including calsequestrin and calnexin, exhibited distinct partitioning between the nucleoplasm and nuclear envelope. These findings will aid in understanding the function of the nuclear calcium pool. 'Second', we investigated nuclear nucleoside triphosphatase (NTPase), an enzyme bound to the nuclear envelope that provides energy for the export of poly A(+) mRNA from the nucleus. Using a novel corpulent rat model, we examined how alterations in nuclear envelope composition affected NTPase activity. Corpulent rats had greatly increased NTPase activity, and this correlated well with an increase in nuclear membrane [cholesterol]. Corpulent rat nuclei also exhibited greater fragility in a novel nuclear membrane integrity assay we developed using salt-induced lysis. Alteration of nuclear envelope composition thus has both structural (nuclear fragility) and functional (NTPase activity) consequences. ' Third', nuclear localization signal-mediated import of proteins to the nucleus through the nuclear pore complex was examined. Many studies have identified cytosolic factors required for nuclear import, however, few have examined import regulation. Using a nuclear protein import assay in permeablized aortic smooth muscle cells, we found that H2O2 inhibited import in a time- and dose dependent manner by acting on a cytosolic factor. H2O2 activated the MAP kinase ERK2. Activated ERK2, in turn, mimicked H2O2's effect on nuclear import. ERK2, therefore, mediates nuclear protein import inhibition caused by H2O2. Immunocytochemis ry revealed that the cytosolic level of Ran, a key import factor, increased specifically in response to H2O2 treatment, which may reflect an alteration in the nucleocytoplasmic cycling of Ran. Altered Ran cycling is known to inhibit import, thus the downstream target of ERK2 may be Ran or one of its accessory proteins. This study was the first to link the MAP kinase pathway with import.