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

项目摘要 核孔复合物（Nuclear pore complex，NPCs）介导蛋白质、RNA和核糖核蛋白的双向转运 复合物穿过真核细胞的双膜核膜。因此，NPC 对于许多生物合成、信号传导和基因调节过程的能力至关重要，以维持细胞健康 和生存能力。由于识别缺陷或NPC结构和功能改变而导致的蛋白质错误定位与 原发性胆汁性肝硬化、肌萎缩侧索硬化（ALS）、白血病和癌症等多种疾病， 以及阿尔茨海默氏症和亨廷顿舞蹈症。而NPC的蛋白质成分和许多可溶性核 已经确定并广泛研究了传输因素，双向传输的机制 发生而不堵塞孔隙仍然是未知的。NPC具有近似八边形对称的 具有沙漏形中心孔的圆柱形结构，内部最小直径约为50-60 nm 在人类身上。堵塞这个孔和装饰其出口是一个网络的> 200移动的内在无序 具有数千个苯丙氨酸-甘氨酸（FG）重复基序的多肽，所述基序为细胞核提供结合位点， 转运受体（NTR），通过NPC携带货物。在稳定状态下，最多约100个NTR 不对称地分布在整个FG网络中。异构动态NTR/FG网络 建立了一个渗透屏障，同时提供了转运输入的途径， 出口各种尺寸、亲和力和表面特性的复合物。多个首选路径通过 存在中心孔。然而，这些结构的重叠性、选择性以及几何和功能性质是不确定的。 易位管道在很大程度上是未开发的，由于历史上缺乏技术工具来解剖这些 具有必要的空间和时间分辨率的路径。为了解决这个问题，一个多色彩的三- 三维（3D）超分辨率荧光显微镜方法是在最后一个赠款期开发的 该系统能够确定与单个功能性NPC相结合的单个功能性NPC的位置和方向， 过境货物的粒子轨迹。这种方法将用于确定结构和功能 多重易位管道和FG网络屏障的特性。具体目标是：1）确定 蛋白质易位管道的数量和性质;和2）确定FG-多肽和NTR FG网络中的分布。目的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个不同的渠道组成，以及是否有任何专门用于进口 或出口。目标2旨在确定FG组分的物理排列和性质如何， 网络与促进所鉴定的易位管道有关。这项工作将直接解决是否 通过渗透性屏障的优选路线提供了避免预期的竞争和堵塞的手段 通过同一通道的双向交通。此外，改进的超分辨率显微镜技术 并且算法被期望包括该领域的必要工具包以及具有广泛的适用性。

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.