The long-term objectives are to understand the role of the nuclear lamina in organization of nuclear envelope and chromosome architecture, the mechanism of nucleocytoplasmic translocation of proteins and RNA across the nuclear pore complex, and the involvement of protein phosphorylation in regulation of mitosis. The functional properties of the lamina will be studied by biochemical and ultrastructural approaches using cell-free systems that yield assembly and disassembly of nuclei in a mitosis-like fashion. A cell-free nuclear assembly system will be reconstituted from isolated components of mitotic tissue culture cells, and will be used to identify discrete steps and intermediates in the process of nuclear envelope assembly and to study the relationship of lamin assembly and dephosphorylation to nuclear envelope formation. Furthermore, different domains of lamins involved in nuclear assembly will be investigated with soluble fragments of isolated lamins and lamin-specific monoclonal antibodies using this cell-free system as well as in vivo a microinjection. A cell-free nuclear disassembly system will be used to isolate the factor that mediates prophase disassembly of the nuclear lamina, which is apparently a mitosis-specific protein kinase. The process of nuclear disassembly will be further analyzed in vivo and in vitro with synthetic peptides containing mitotic lamin phosphorylation sites. Monoclonal antibodies will be prepared to new polypeptides and structural domains of the nuclear pore complex, and in combination with a battery of pore-specific monoclonal antibodies that have already been obtained, will be used to dissect the substructural organization of the pore complex by fractionation of mitotic cells and analysis of in vitro pore complex assembly. These antibodies will also be used to define involvement of specific pore complex components in different types of nucleocytoplasmic transport transport by in vivo microinjection. A cell-free system to study transport of large proteins into the nucleus will be devised, and analyzed with biochemical approaches and monoclonal antibodies to define the components and steps involved in active transport of proteins across the nuclear pore complex.
NIH ExPorter
NIH ExPorter