The Nuclear Pore Complex: Interphase and Mitotic Function

描述(申请人提供)：细胞分裂过程中基因组内容的准确遗传依赖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，一旦染色体发生凝聚，核膜、核孔复合体和核层就会一致解体。这个项目的长期目标是阐明有丝分裂事件是如何被编排的。核孔蛋白功能和细胞周期之间的界面是这个谜题中一个新出现的和关键的部分。在这里，我们将探讨最近发现的核孔蛋白Nup153在有丝分裂中的作用。有丝分裂解体的机制也将被研究，以及有丝分裂结束时拆解如何与核重组联系在一起。具体地说，在目标1中，将利用哺乳动物细胞中Nup153的敲除结合结构-功能挽救分析来破译Nup153在有丝分裂早期和晚期的功能。为了跟踪哪些特定的有丝分裂标志被改变，细胞分裂事件将通过实时成像进行监测。通过候选和不偏不倚的方法确定相关的蛋白质伙伴，将用于进一步磨练Nup153的贡献。第二个目的是研究苏莫化在调节Nup153功能中的作用。将绘制苏莫化位点(S)的图谱，并将在击倒救援实验中以及针对孔结构的特定特征来测试干扰这一修饰的功能后果。最后，在第三个目标中，在无细胞非洲爪蛙卵提取系统中重组的细胞核将被用作工具来研究小GTP酶Arf的作用以及它作为一个节点刺激磷脂酶D的能力，该节点为膜和膜的分解提供整合信号。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被膜重组的膜的作用将被测试。这项研究将对细胞分裂的事件以及它们是如何协调的提供新的见解。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编排的。当这种协调被打乱时，细胞核就不能正确地重塑。这种形态和DNA含量的缺陷是侵袭性癌细胞的特征，这里提出的研究将有助于更好地理解这些变化是如何发生的。

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.