Mechanisms of Nucleocytoplasmic Transport

描述（由申请人提供）：核孔复合物（NPCs）介导蛋白质、rna和核糖核蛋白复合物在真核细胞双膜核包膜上的双向运输。因此，适当的NPC功能对于各种细胞生物合成和调节过程至关重要。NPC结构和功能特性的改变与各种人类疾病有关，包括白血病、癌症和原发性胆汁性肝硬化。通过npc进行的分子交易是高度调控和微妙平衡的；在细胞质和核质间穿梭的单一基因调节因子（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病也可能与核转运有关。虽然鼻咽癌本身的许多蛋白质成分和许多可溶性蛋白质辅助因子已经被确定和广泛研究，但孔选择性和货物通过鼻咽癌的分子机制仍然很大程度上未知。为了进一步研究核胞质转运的基本特征，研究人员开发了单分子荧光（SMF）显微镜和单粒子跟踪技术，以高达1 ms的时间分辨率直接观察通过npc的分子运输。这种方法可以直接测量货物转运时间和进口效率，并可以表征NPC内货物流动的各个方面。令人惊讶的是，在体外，通过改变输入蛋白2的浓度，转运的Vmax可以改变至少10倍。目前尚不清楚细胞在多大程度上利用这一机制根据需要积极调节核贩运率。提出的研究目标是通过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)描述Imp ？/CAS在核进口时进行综合装配，在核出口时进行拆卸；(2)确定运输路径重叠对信号依赖型和非信号依赖型货物转运时间和进口效率的影响；(3)确定小恶魔的数量？在体内稳态和在渗透细胞中分离的npc中的辅助因子；(4)确定核定位序列数量对货物在FG-Nup网络内的交互频率、转运时间、进口效率和平均分布的影响。这些实验旨在探索npc通过不同途径运输各种货物时所享有的广泛参数空间，期望它们将从根本上促进我们对核胞质运输各种机制的理解。公共卫生相关性：由于核孔复合物（NPCs）为细胞质和细胞核之间的重要物质和信息传递提供了一个焦点，核细胞质运输系统的功能障碍对细胞的健康和活力有严重的影响。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化有关，还可能与阿尔茨海默氏症和亨廷顿氏病有关。核细胞质转运的基本生化机制将被表征，以便未来的研究可以建立在对转运如何维持或通过功能障碍无法维持细胞和组织中的代谢调节和组织的坚定理解之上。

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.