PhD

dissertationIn higher eukaryotes, each time a cell divides dramatic changes occur at the nuclear periphery. The nuclear envelope, nuclear pore complexes, and nuclear lamina must disassemble to allow the mitotic spindle access to the genomic DNA, but little is known about the mechanisms required for these breakdown events. In order to biochemically dissect these events, we have optimized an assay to study nuclear disassembly in vitro using Xenopus egg extracts. One of the early events in nuclear envelope breakdown is disassembly of the nuclear pore complexes suggesting that the nuclear pores may be an important organizing center of mitotic events. We have identified a role for two nuclear pore proteins, Nup153 and Nup358/RanBP2, in recruiting the COPI coatomer complex to the nuclear envelope during nuclear disassembly. Adding antibody specific to either of these nuclear pore proteins individually inhibits nuclear disassembly, suggesting the nucleoporins are required in a nonredundant manner. The zinc finger domains within these nucleoporins appear to be the critical determinants for their role in nuclear envelope breakdown, since addition of recombinant zinc finger fragments derived from either pore protein alters COPI recruitment to the nuclear envelope and interferes with nuclear envelope breakdown. The localization of Nup153 and Nup358 to the nuclear basket and cytoplasmic filaments of the nuclear pore, respectively, suggests that recruitment of COPI to both faces of the pore is important. The COPI complex is known to have a role in vesicle trafficking from the Golgi to the ER and may play a similar role at the nuclear envelope during disassembly. The zinc finger fragments also prevent disassembly of the nuclear lamina, suggesting that recruitment of COPI to the nuclear envelope may be critical for coordinating dispersal of the membrane with other cell cycle events. Nuclei disassembled during prophase must then be efficiently reassembled during anaphase. Using dominant negative recombinant protein fragments, we have found that Nup153 interacts with regulators required for nuclear assembly and DNA replication. These results provide new information about the critical components of nuclear assembly and disassembly and lay the foundation for a better understanding of how these processes are regulated