Studies of proliferating cell nuclear antigen (PCNA) and fibroblast growth factor receptor-1 (FGFR-1) in the mammalian cell nucleus

Recent studies indicate a spatio-temporal organization of nuclear functions, such as replication, transcription and RNA splicing within the three dimensional space of the nucleus. Factors and enzymes involved in these processes are known to target to the respective sites of function within the nucleus. Thus, the spatial organization of proteins within the nucleus is of fundamental importance in analyzing their role in nuclear function. In addition, all known nuclear functions are also maintained on the isolated nuclear matrix—an underlying structural component within the nucleus. The analysis of the spatio-temporal organization of two essential proteins, proliferating cell nuclear antigen (PCNA) and fibroblast growth factor receptor 1 (FGFR-1) within the nuclei of mammalian cells forms the foundation of this thesis.; PCNA is an essential replication protein involved in replication fork elongation. Using laser scanning confocal microscopy and computer image analysis we demonstrate that PCNA associates with sites of replication throughout S-phase. In addition, PCNA dissociates from replication sites upon completion of DNA synthesis and translocates to new sites of replication. The dynamics of PCNA movement during S-phase were monitored in real time using a stably transfected GFP-PCNA construct under the regulated control of a tetracycline inducible promoter. We observed positional changes in individual replication sites imaged within short intervals of time during early-S and late-S phase.; In contrast to PCNA, FGFR-1 belongs to a family of tyrosine kinase receptors normally found on the surface of cells. Recent data, however, indicate the presence of FGFR-1 within the nucleus of cells. In this report we demonstrate that a constitutive form of FGFR-1 is present in the nuclei of rapidly proliferating medulloblastoma cells and strongly colocalizes with sites of splicing factor rich nuclear speckles. In addition, moderate colocalization was also observed between sites of FGFR-1 and transcription. However, no colocalization was observed with sites of replication.; Both FGFR-1 and PCNA were maintained on isolated nuclear matrices. This strongly suggests a multifunctional role for this nuclear substructure in organizing and moderating various nuclear functions. The studies reported in this dissertation, though focusing on two very different nuclear proteins, addresses fundamental questions about the relationship of structure and function within the nucleus of mammalian cells.