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

使用 3D 超分辨率显微镜绘制通过核孔的传输路径

基本信息

批准号：
10707468
负责人：
SIEGFRIED M MUSSER
金额：
$ 31.47万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10707468
关键词：
3-Dimensional Address Affinity Algorithms Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Behavior Binding Binding Sites Biochemical Biocompatible Materials Biological Biophysics Carrier Proteins Cell Nucleus Cell physiology Cells Central Pore of the Nuclear Pore Complex Chickens Color Complex Cytoplasm Dedications Defect Devices Diameter Diffusion Disease Environment Eukaryotic Cell Filtration Fluorescence Microscopy Foundations Functional disorder Gatekeeping Gene Expression Glycine Goals Grant Growth Health Heterogeneity Human Huntington Disease Imaging technology Individual Infrastructure Link Location Malignant Neoplasms Maps Mediating Membrane Proteins Modeling Nature Nuclear Envelope Nuclear Pore Nuclear Pore Complex Nucleic Acids Nucleoplasm Outcome Pathway interactions Pattern Peripheral Permeability Phenylalanine Play Positioning Attribute Primary biliary cirrhosis Process Property Protein translocation Proteins RNA Regulator Genes Research Resolution Ribonucleoproteins Role Route Shapes Signal Transduction Signal Transduction Pathway Structure Surface Properties System Technology Testing Three-Dimensional Imaging Time Tissues Transportation Visualization Work density design flexibility human disease imaging approach improved insight leukemia macromolecule migration millisecond nanomechanics nanoscale new technology nucleocytoplasmic transport particle polypeptide preference protein transport receptor response scaffold single molecule small molecule sound superresolution imaging superresolution microscopy temporal measurement tool ultra high resolution

项目摘要

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.

项目摘要核孔复合物（NPC）介导蛋白质，RNA和核糖核蛋白的双向转运跨真核细胞的双膜核包膜的复合物。因此，NPC是许多生物合成，信号传导和基因调节过程保持细胞健康的能力至关重要和生存能力。由于识别缺陷或变化的NPC结构和功能而引起的蛋白质错误定位是链接的像原发性胆汁肝硬化一样多样化的疾病，肌萎缩性侧索硬化症（ALS），白血病和癌症，还有阿尔茨海默氏症和亨廷顿的疾病。而NPC的蛋白质成分和许多可溶性核成分已经确定并广泛研究了运输因子，该机制是双向转运的机制发生而不会堵塞孔仍然未知。 NPC的八角形大约对称圆柱结构，带有沙漏形的中央孔，内部最小直径约为50-60 nm 在人类中。阻塞此孔并装饰其出口是一个> 200个移动杂物的网络多肽具有数千种苯丙氨酸 - 甘氨酸（FG）重复基序，可为核提供结合位点运输受体（NTR），这些受体通过NPC携带碳。在稳定状态下，最多约100个NTR是不对称分布在整个FG网络中。异构和动态的NTR/FG网络建立一个渗透性障碍，同时为进口易位提供途径各种大小，亲和力和表面特性的出口复合物。多个首选路径中心孔存在。但是，这些重叠，选择性和几何和功能特性易位导管在很大程度上没有探索，因为缺乏技术工具来剖析这些工具具有必要的空间和时间分辨率的途径。为了解决这一缺陷，多色三尺寸（3D）超分辨率荧光显微镜方法是在最后一个赠款期间开发的能够确定单个功能NPC与单个功能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个易位导管和FG网络屏障的性质。具体目的是：1）确定蛋白质转运导管的数量和性质； 2）确定FG-溶木和NTR FG网络中的分布。 AIM 1试图探索至少三个不同的易位的可能性导管，其中一些是否由8个不同的渠道组成，以及任何专门用于进口的渠道或出口。 AIM 2试图确定FG-组件的物理排列和特性如何网络与促进已确定的易位管道有关。这项工作将直接解决是否通过渗透性屏障的首选路线提供了一种避免竞争和堵塞预期的手段对于通过同一频道的双向流量。此外，改进的超分辨率显微镜技术预计算法将构成该领域的必要工具包，并具有广泛的适用性。