Accurate inheritance of genomic content during cell division is dependent on synchronized changes in cellular organization and chromosome dynamics. In higher eukaryotes, once chromosomes have undergone condensation, the nuclear membranes, nuclear pore complexes, and nuclear lamina are disassembled in concert. The long-term goal of this project is to elucidate how events of mitosis are orchestrated. The interface between nuclear pore protein function and the cell cycle is an emerging and critical piece of this puzzle. Here, recently identified roles of the nucleoporin Nup153 at mitosis will be pursued. Mechanisms that integrate events of mitotic disassembly will also be investigated, as well as how disassembly is connected with nuclear reassembly at the end of mitosis. Specifically, in Aim 1, knockdown of Nup153 in mammalian cells in combination with a structure-function rescue analysis will be employed to decipher the functions of Nup153 in both early and late mitosis. In order to track which specific hallmarks of mitosis are altered, events of cell division will be monitored by live imaging. Identification of relevant protein partners, through both candidate and unbiased approaches, will be used to further hone in on the contributions of Nup153.
The second Aim i s focused on characterizing the role of sumoylation in regulating Nup153 function. The site(s) of sumoylation will be mapped and the functional consequence of interfering with this modification will be tested, both in knockdown-rescue experiments as well as with respect to specific features of pore architecture. Finally, in the third Aim, nuclei reconstituted in the cell-free Xenopus egg extract system will be used as a tool to investigate the role of the small GTPase Arf and its ability to stimulate phospholipase D as a node that provides an integrative signal for lamina and membrane disassembly. A role for COPI in organizing membrane populations at mitosis and how this affects the consequent participation of membranes in reassembly of the nuclear envelope will be tested. This research will yield new insight into the events of cell division and how they are coordinated. In turn, this information will impact our understanding of how abnormal nuclear morphology and DNA content arise, which is an imperative step in deciphering the molecular events that lead to cancerous cell proliferation.
The goals of this research are to better understand how the events of cell division are orchestrated. When this coordination is disrupted, the cell nucleus does not reform properly. Such defects in morphology and DNA content are hallmarks of aggressive cancer cells, and the research proposed here will contribute to a better understanding of how these alterations arise.
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