Mechanisms of Nucleocytoplasmic Transport

核质运输机制

• 批准号: 7849508
• 负责人: SIEGFRIED M MUSSER
• 金额: $ 34.79万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7849508
• 关键词: Address; Algorithms; Alzheimer' s Disease; Binding; Biochemical; Carrier Proteins; Cell Nucleus; Cell Survival; Cells; Characteristics; Complex; Couples; Cytoplasm; Data; Diffusion; Dissociation; Equilibrium; Eukaryotic Cell; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Frequencies; Functional disorder; Future; GTP-Binding Proteins; Gene Expression; Goals; Guanosine Triphosphate; Health; Huntington Disease; Hydrolysis; Imagery; In Vitro; Individual; Investigation; Knowledge; Link; Malignant Neoplasms; Measurement; Mediating; Membrane; Metabolic; Modeling; Molecular; Motor; Movement; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Import; Nuclear Pore Complex; Nucleic Acids; Nucleoplasm; Pathway interactions; Permeability; Primary biliary cirrhosis; Process; Property; Proteins; Regulation; Regulator Genes; Research; Resolution; Ribonucleoproteins; Ribosomes; Running; Signal Transduction; Signal Transduction Pathway; Speed; Structure; System; Techniques; Testing; Time; Tissues; Tumor Suppressor Proteins; cell growth; cofactor; computerized data processing; design; driving force; expectation; human disease; in vivo; leukemia; nucleocytoplasmic transport; particle; physical property; protein transport; public health relevance; research study; response; single molecule; small molecule; trafficking

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.

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的双向转运，横跨真核细胞的双膜核包膜。因此，适当的NPC功能对于多种细胞生物合成和调节过程至关重要。 NPC的结构和功能性质改变与包括白血病，癌症和原发性胆道肝硬化在内的各种人类疾病有关。通过NPC进行的分子运输受到高度调节和精致的平衡。单个基因调节因子的效率低下或过度转运，该因子在细胞质和核质室之间穿梭，例如肿瘤抑制剂，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运输有关。尽管已经鉴定出和广泛研究了NPC本身的许多蛋白质成分以及许多可溶性蛋白辅因子，但孔选择性和通过NPC的货物通道的分子机制仍然在很大程度上未知。为了进一步检查核细胞质转运的基本特征，开发了单分子荧光（SMF）显微镜和单粒子跟踪技术，以直接观察到通过NPC的分子运输，最多1 ms。这种方法允许直接测量货物易位时间及其进口效率，并允许表征NPC内货物运动的各个方面。出乎意料的是，通过在体外改变imptimin 2浓度，可以改变运输的VMAX至少10倍。目前尚不清楚细胞利用该机制积极调节核贩运率以响应需求的程度。拟议的研究的目标是通过SMF显微镜从根本上促进我们对NPC功能的了解。该项目的具体目的是：（1）表征核进口期间的IMP？/cas复合物组件； （2）确定传输途径对信号依赖性和非依赖性cargos的易位时间和进口效率的影响； （3）确定IMP的数量？ NPC在体内稳态的NPC中的辅因子，并在通透性细胞中分离； （4）确定核定位序列数量对FG-NUP网络中货物相互作用频率，易位时间，进口效率和平均分布的影响。这些实验旨在探索NPC通过不同的途径运输各种cargos所享有的广泛参数空间，并希望它们从根本上可以促进我们对核质交通的各种机制的理解。公共卫生相关性：由于核孔复合物（NPC）为细胞质和核之间必不可少的材料和信息的继电器提供了一个焦点，因此核质质运输系统的功能障碍对细胞的健康和生存能力产生了严重的后果。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，可能与阿尔茨海默氏症和亨廷顿疾病有关。将表征核质转运的基本生化机制，以便可以基于对转运如何维持或通过功能障碍的坚定理解，无法维持，在细胞和组织中的代谢调节和组织。