The Nuclear Pore Complex: Interphase and Mitotic Function

描述（由申请人提供）：细胞分裂期间基因组内容的准确遗传取决于细胞组织和染色体动力学的同步变化。在高等真核生物中，一旦染色体发生浓缩，核膜、核孔复合体和核纤层就一起解体。这个项目的长期目标是阐明有丝分裂事件是如何被精心安排的。核孔蛋白功能与细胞周期之间的界面是这个难题中一个新兴且关键的部分。在这里，最近确定的核孔蛋白Nup 153在有丝分裂中的作用将继续进行。整合有丝分裂解体事件的机制也将被研究，以及解体如何与有丝分裂结束时的核重组联系在一起。具体而言，在目标1中，哺乳动物细胞中Nup 153的敲低结合结构-功能拯救分析将用于破译Nup 153在早期和晚期有丝分裂中的功能。为了跟踪有丝分裂的哪些特定标志被改变，将通过实时成像监测细胞分裂事件。通过候选和无偏见的方法鉴定相关蛋白质伴侣，将用于进一步研究Nup 153的贡献。第二个目的是集中在表征类小泛素化在调节Nup 153功能中的作用。将绘制类小泛素化的位点，并在敲除-拯救实验中以及相对于孔结构的特定特征测试干扰该修饰的功能后果。最后，在第三个目的，核重建的无细胞非洲爪蟾卵提取物系统将被用作一种工具，以调查的作用，小GTdR Arf和它的能力，刺激磷脂酶D作为一个节点，提供了一个综合信号的薄层和膜拆卸。COPI在有丝分裂时组织膜群体的作用以及这如何影响随后参与核膜重组的膜将被测试。这项研究将对细胞分裂事件以及它们如何协调产生新的见解。反过来，这些信息将影响我们对异常核形态和DNA含量如何产生的理解，这是破译导致癌细胞增殖的分子事件的必要步骤。公共卫生相关性：这项研究的目标是更好地了解细胞分裂事件是如何精心策划的。当这种协调被破坏时，细胞核就不能正确地改革。这种形态和DNA含量的缺陷是侵袭性癌细胞的标志，这里提出的研究将有助于更好地理解这些改变是如何产生的。

DESCRIPTION (provided by applicant): 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 is 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. PUBLIC HEALTH RELEVANCE: 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.