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穿过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至关重要。 NPC 结构和功能特性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化。通过 NPC 的分子贩运受到高度监管和微妙的平衡；在细胞质和核质区室之间穿梭的单个基因调节因子（例如肿瘤抑制因子）的低效或过量运输与多种癌症有关。阿尔茨海默病和亨廷顿病也可能与核运输有关。虽然 NPC 本身的许多蛋白质成分和许多可溶性蛋白质辅助因子已被鉴定并进行了广泛研究，但孔选择性和货物通过 NPC 的分子机制仍然很大程度上未知。为了进一步研究核细胞质运输的基本特征，开发了单分子荧光 (SMF) 显微镜和单粒子跟踪技术，以高达 1 毫秒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及其进口效率，并可以描述 NPC 内货物移动的各个方面。令人惊讶的是，通过改变体外导入蛋白 2 的浓度，转运的 Vmax 可以改变至少约 10 倍。目前尚不清楚细胞在多大程度上利用这种机制来主动调节核运输速率以响应需要。本研究的目标是通过 SMF 显微镜从根本上增进我们对 NPC 功能的了解。该项目的具体目标是： (1) 表征核进口过程中的Imp ?/CAS复杂组装和核出口过程中的拆卸； (2) 确定运输路径重叠对信号依赖型和信号无关型货物的转运时间和进口效率的影响； (3) 确定Imp 的数量？体内稳态和透化细胞中分离的 NPC 中的辅因子； (4)确定核定位序列的数量对货物在FG-Nup网络内的相互作用频率、易位时间、输入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增进我们对核细胞质运输各种机制的理解。公共健康相关性：由于核孔复合体 (NPC) 提供了细胞质和细胞核之间重要物质和信息传递的焦点，因此核细胞质运输系统的功能障碍会对细胞的健康和活力产生严重后果。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，还可能与阿尔茨海默病和亨廷顿病有关。核细胞质运输的基本生化机制将被表征，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无法维持细胞和组织中的代谢调节和组织的牢固理解的基础上。