Mechanisms of Nucleocytoplasmic Transport

描述（由申请人提供）：核孔复合物（NPC）介导蛋白质、RNA和核糖核蛋白复合物跨真核细胞双膜核膜的双向转运。因此，适当的NPC功能对于多种细胞生物合成和调节过程是必不可少的。NPC的结构和功能特性的改变与包括白血病、癌症和原发性胆汁性肝硬化在内的各种人类疾病有关。通过NPC的分子运输是高度调节和微妙平衡的;在细胞质和核质区室之间穿梭的单个基因调节因子（如肿瘤抑制因子）的低效或过量运输与各种癌症相关。阿尔茨海默氏症和亨廷顿氏病也可能与核运输有关。虽然已经鉴定和广泛研究了NPC本身的许多蛋白质组分和许多可溶性蛋白质辅因子，但是孔选择性和货物通过NPC的分子机制在很大程度上仍然未知。为了进一步研究核质转运的基本特征，开发了单分子荧光（SMF）显微镜和单粒子跟踪技术，以直接观察分子通过NPC的运输，时间分辨率高达1 ms。这种方法允许直接测量货物转运时间及其进口效率，并允许表征NPC内货物流动的各个方面。令人惊讶的是，通过改变体外输入蛋白2浓度，转运的Vmax可以改变至少~10倍。目前尚不清楚，这些组织在多大程度上利用这一机制，根据需要积极调节核贩运率。拟议研究的目标是通过SMF显微镜从根本上提高我们对NPC功能的认识。该项目的具体目的是：（1）表征Imp？/ CAS复合物组装过程中的核输入和拆卸过程中的核输出;（2）确定转运途径重叠的转运时间和输入效率的信号依赖和非依赖的货物的影响;（3）确定Imp？（4）确定核定位序列的数目对货物的相互作用频率、易位时间、输入效率和FG-Nup网络内的平均分布的影响。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质运输各种机制的理解。公共卫生相关性：由于核孔复合物（nuclear pore complex，NPC）为细胞质和细胞核之间的必需物质和信息的传递提供了一个焦点，因此核质转运系统的功能障碍对细胞的健康和活力具有严重的后果。例如，NPC的结构和功能与白血病、癌症和原发性胆汁性肝硬化有关，并且可能与阿尔茨海默病和亨廷顿病有关。核质运输的基本生化机制的特点，使未来的调查可以建立在一个坚定的理解如何运输维护，或通过功能障碍未能维持，代谢调节和组织在细胞和组织。

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.