Mechanisms of Nucleocytoplasmic Transport

描述(申请人提供)：核孔复合体(NPC)介导蛋白质、RNA和核糖核蛋白复合体通过真核细胞的双膜核膜的双向运输。因此，适当的NPC功能对于各种细胞生物合成和调节过程是必不可少的。鼻咽癌结构和功能的改变与多种人类疾病有关，包括白血病、癌症和原发性胆汁性肝硬变。通过核祖细胞的分子运输受到高度调控和微妙的平衡；穿梭于细胞质和核质之间的单一基因调控因子(如肿瘤抑制因子)的低效或过度运输与各种癌症有关。阿尔茨海默氏症和亨廷顿氏症也可能与核运输有关。虽然鼻咽癌本身的许多蛋白质组分和许多可溶性蛋白辅助因子已经被鉴定和广泛研究，但孔选择性和货物通过鼻咽癌的分子机制仍然很不清楚。为了进一步研究核质运输的基本特征，发展了单分子荧光显微镜(SMF)和单粒子跟踪技术，以高达1ms的时间分辨率直接观察通过核祖细胞的分子运输。这种方法可以直接测量货物转运时间及其进口效率，并可以描述全国人大内货物运输的各个方面。令人惊讶的是，通过改变Importin 2的体外浓度，转运的Vmax至少可以改变~10倍。目前尚不清楚细胞在多大程度上利用这一机制积极管制核贩运比率，以应对需要。本研究的目的是通过SMF显微镜从根本上提高我们对鼻咽癌功能的认识。该项目的具体目标是：(1)确定核进口和核出口拆解过程中Imp？/CAS复合体组装的特征；(2)确定运输路径重叠对信号依赖和非独立货物的转运时间和进口效率的影响；(3)确定Imp？/CAS的数量。(4)确定核定位序列的数量对货物的相互作用频率、转运时间、进口效率和在FG-NUP网络中的平均分布的影响。这些实验旨在探索NPC通过不同的路径运输各种货物时所享有的广泛的参数空间，期望它们将从根本上促进我们对核质运输各种机制的理解。与公共卫生的相关性：由于核孔复合体为细胞质和细胞核之间的基本物质和信息传递提供了一个焦点，核质运输系统的功能障碍对细胞的健康和生存能力具有严重的后果。例如，鼻咽癌的结构和功能与白血病、癌症和原发性胆汁性肝硬变有关，还可能与阿尔茨海默氏症和亨廷顿氏病有关。核质运输的基本生化机制将被描述，以便未来的研究可以建立在对运输如何维持或通过功能障碍维持细胞和组织中的代谢调节和组织的坚定理解的基础上。

DESCRIPTION (provided by applicant): Nuclear pore complexes (NPCs) mediate the bidirectional transport of proteins, RNAs and ribonucleoprotein complexes across the double-membrane nuclear envelope of eukaryotic cells. Consequently, proper NPC function is essential for a wide variety of cellular biosynthetic and regulatory processes. Altered structural and functional properties of the NPC are linked with various human diseases including leukemias, cancers and primary biliary cirrhosis. The molecular trafficking through NPCs is highly regulated and delicately balanced; inefficient or excess transport of a single gene regulatory factor that shuttles between cytoplasmic and nucleoplasmic compartments, such as a tumor suppressor, is associated with various cancers. Alzheimer's and Huntington's disease may also be linked to nuclear transport. While many protein components of the NPC itself and many soluble protein cofactors have been identified and extensively studied, the molecular mechanisms of pore selectivity and of cargo passage through the NPC remain largely unknown. To further examine the fundamental characteristics of nucleocytoplasmic transport, single molecule fluorescence (SMF) microscopy and single particle tracking techniques were developed to directly observe molecules trafficking through NPCs with up to 1 ms time resolution. This approach allows direct measurement of cargo translocation times and their import efficiencies, and allows characterization of various aspects of cargo movement within the NPC. Surprisingly, the Vmax for transport can be altered at least ~10-fold by changing the importin 2 concentration in vitro. It remains unclear the extent to which cells utilizes this mechanism to actively regulate nuclear trafficking rates in response to need. The goals of the proposed research are to fundamentally advance our knowledge of NPC function via SMF microscopy. The Specific Aims of the project are: (1) to characterize Imp ?/CAS complex assembly during nuclear import and disassembly during nuclear export; (2) to determine the effect of transport pathway overlap on the translocation time and import efficiency of signal-dependent and -independent cargos; (3) to determine the number of Imp ? cofactors in NPCs at steady-state in vivo and as-isolated in permeabilized cells; and (4) to determine the effects of the number of nuclear localization sequences on a cargo's interaction frequency, translocation time, import efficiency and average distribution within the FG-Nup network. These experiments are designed to explore the wide parameter space enjoyed by NPCs as they transport a variety of cargos by distinct pathways, with the expectation that they will fundamentally advance our understanding of various mechanisms of nucleocytoplasmic transport. Public Health Relevance: Since nuclear pore complexes (NPCs) provide a focal point for the relay of essential materials and information between the cytoplasm and nucleus, dysfunction of the nucleocytoplasmic transport system has grave consequences for the health and viability of the cell. For example, NPC structure and function has been linked to leukemias, cancers and primary biliary cirrhosis, and possibly to Alzheimer's and Huntington's diseases. The basic biochemical mechanisms of nucleocytoplasmic transport will be characterized so that future investigations can be founded on a firm understanding of how transport maintains, or through dysfunction fails to maintain, metabolic regulation and organization in cells and tissues.