The Nuclear Pore Complex: Interphase and Mitotic Function

描述（由申请人提供）：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、核孔复合物和核层就会一起解体。这个项目的长期目标是阐明有丝分裂的事件是如何安排的。核孔蛋白功能和细胞周期之间的界面是这个谜题的一个新兴和关键的部分。本文将探讨最近发现的核孔蛋白Nup153在有丝分裂中的作用。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。具体来说，在Aim 1中，哺乳动物细胞中Nup153的敲低结合结构-功能拯救分析将被用来破译Nup153在早期和晚期有丝分裂中的功能。为了追踪有丝分裂的哪些特定标志被改变，细胞分裂的事件将被实时成像监测。通过候选方法和无偏方法鉴定相关蛋白伴侣，将用于进一步研究Nup153的贡献。第二个目标集中在描述sumoylation在调节Nup153功能中的作用。sumoylation的位点将被绘制出来，干扰这种修饰的功能后果将被测试，这将在敲除拯救实验中进行，也将在孔结构的特定特征方面进行测试。最后，在第三篇论文中，我们将利用在无细胞爪蟾卵提取系统中重建的细胞核作为工具，研究小GTPase Arf的作用及其刺激磷脂酶D作为一个节点的能力，该节点为层和膜的分解提供了一个综合信号。在有丝分裂中，COPI在组织膜种群中的作用，以及这如何影响膜在核膜重组中的后续参与，将被测试。这项研究将对细胞分裂事件及其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：本研究的目的是更好地了解细胞分裂事件是如何被精心安排的。当这种协调被破坏时，细胞核就不能正常重组。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.