| Protein C4 (PC4) is a highly conserved polypeptide doublet of Mr ? 21kD, associated with actin filaments in many cell types. The higher molecular weight, oncogenically down-regulated isoform (transgelin) has been purified and functionally characterised and in vitro has been shown to gelate actin (Shapland et al., 1988). The lower molecular weight isoform (C41) has not yet been functionally defined. Previous immunofluorescence data using an anti-PC4 monoclonal antibody has indicated the presence of PC4 in fibroblast nuclei. Furthermore, DNA sequence data has shown that C41 has a putative Nuclear Localisation Signal. These observations led to this study on nuclear PC4, and to the confirmation of the presence of both isoforms of PC4 in the nucleus of Rat Embryo Fibroblasts by Western immunoblotting. Following this, cells were arrested in various stages of the cell cycle by metabolic inhibitors and variations in culture conditions. This work determined levels of expression of PC4 in the nucleus and in the cytoplasm at defined stages of the cell cycle, indicating a down-regulation of C41 in late G1 and S phases. This data was complemented by FACS (Fluorescence Activated Cell Sorter) analysis which determines the percentage of cells in each phase of the cell cycle, and thus confirms the actions of inhibitors. Further studies on the properties of nuclear PC4 were carried out as attempts were made to solubilise the protein from the nucleus. Nuclease digestion and varying ionic conditions techniques were used to try to partially digest the internal structure of the nucleus, thus attempting to define which molecule(s) PC4 interacts with in the nucleus. |
