Mapping Transport Pathways through Nuclear Pores using 3D Super-Resolution Microscopy

项目总结 核孔复合体(NPC)介导蛋白质、RNA和核糖核蛋白的双向运输 跨越真核细胞双膜核膜的复合体。因此，NPC是 对许多生物合成、信号和基因调控过程维持细胞健康的能力至关重要 和生存能力。由于识别缺陷或鼻咽癌结构和功能改变而导致的蛋白质错误定位 到各种疾病，如原发性胆汁性肝硬变、肌萎缩侧索硬化症、白血病和癌症， 阿尔茨海默氏症和亨廷顿氏症。而鼻咽癌的蛋白质成分和许多可溶性核 运输因素已经确定并被广泛研究，双向运输的机制(S) 在没有堵塞毛孔的情况下发生，目前仍不清楚。NPC有一个八角形对称的近似 具有沙漏状中心孔的圆柱形结构，其内部最小直径约为50-60 nm 在人类身上。堵塞这个毛孔并装饰它的出口的是一个本质上无序的&gt；200手机网络 含有数千个苯丙氨酸-甘氨酸(FG)重复基序的多肽为核提供结合部位 运送货物通过NPC的运输受体(NTR)。在稳定状态下，多达~100个NTR 不对称地分布在整个FG网络中。异构型动态NTR/FG网络 建立渗透屏障，同时为进口和进口货物的转移提供途径 出口各种尺寸、亲和力和表面性质的复合体。多条首选路径通过 中央孔道存在。然而，它们的重叠性、选择性以及几何和功能属性 由于历史上缺乏分析这些通道的技术工具，转运管道在很大程度上还没有被探索。 具有必要的空间和时间分辨率的路径。为了解决这一不足，一款多色三色- 三维(3D)超分辨荧光显微镜方法是在最后一次赠款期间开发的 能够确定单个功能NPC与单个 过境货物的粒子轨迹。这种方法将用于确定结构和功能 多个易位管道的特性和FG网络屏障。具体目标是：1)确定 蛋白质转运管道的数量和性质；2)测定FG多肽和NTR FG网络内的分布。目标1试图探索至少三种不同易位的可能性 管道，其中一些是否由8个不同的通道组成，以及是否有专用于任一导入的通道 或者出口。目标2试图确定FG组件的物理排列和性质如何- 网络与促进已确定的转运管道相联系。这项工作将直接解决是否 通过渗透屏障的首选路线提供了一种避免预期的竞争和堵塞的方法 用于通过相同通道的双向流量。此外，改进的超分辨率显微镜技术 预计算法将包括该领域的必要工具包，并具有广泛的适用性。

PROJECT SUMMARY Nuclear pore complexes (NPCs) mediate the bi-directional transport of proteins, RNAs and ribonucleoprotein complexes across the double-membrane nuclear envelope of eukaryotic cells. Consequently, NPCs are essential for the ability of many biosynthetic, signaling and gene regulatory processes to maintain cellular health and viability. Protein mis-localization due to recognition defects or altered NPC structure and function is linked to diseases as diverse as primary biliary cirrhosis, amyotrophic lateral sclerosis (ALS), leukemias and cancers, and Alzheimer's and Huntington's diseases. While the protein components of the NPC and many soluble nuclear transport factors have been identified and extensively studied, the mechanism(s) by which bi-directional transport occurs without clogging the pore remains unknown. The NPC has an octagonally symmetric approximately cylindrical structure with an hourglass-shaped central pore that has an internal minimal diameter of ~50-60 nm in humans. Occluding this pore and decorating its exits is a network of > 200 mobile intrinsically disordered polypeptides with thousands of phenylalanine-glycine (FG) repeat motifs that provide binding sites for the nuclear transport receptors (NTRs) that carry cargos through the NPC. At steady-state, up to ~100 NTRs are asymmetrically distributed throughout this FG-network. The heterogeneous and dynamic NTR/FG-network establishes a permeability barrier while simultaneously providing pathways for the translocation of import and export complexes of a wide range of sizes, affinities and surface properties. Multiple preferred paths through the central pore exist. However, the overlap, selectivity, and geometric and functional properties of these translocation conduits are largely unexplored due to the historical absence of technological tools to dissect these pathways with the necessary spatial and temporal resolution. To address this deficiency, a multi-color three- dimensional (3D) super-resolution fluorescence microscopy approach was developed in the last grant period that is capable of determining the position and orientation of individual functional NPCs combined with single particle trajectories of transiting cargo. This approach will be used to determine the structural and functional properties of multiple translocation conduits and the FG-network barrier. The Specific Aims are: 1) to determine the number and nature of protein translocation conduits; and 2) to determine the FG-polypeptide and NTR distributions within the FG-network. Aim 1 seeks to explore the possibility of at least three distinct translocation conduits, whether some of these consist of 8 distinct channels, and whether any are dedicated to either import or export. Aim 2 seeks to determine how the physical arrangement and properties of components of the FG- network are linked to promoting the identified translocation conduits. This work will directly address whether preferred routes through the permeability barrier provide a means to avoid the competition and clogging expected for two-way traffic through the same channel. In addition, improved super-resolution microscopy technologies and algorithms are expected to comprise a necessary toolkit for the field as well as to have broad applicability.