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.

项目概要核孔复合物 (NPC) 介导蛋白质、RNA 和核糖核蛋白的双向转运跨真核细胞双膜核膜的复合物。因此，NPC 对于许多生物合成、信号传导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 NPC 结构和功能改变而导致的蛋白质错误定位是相关的原发性胆汁性肝硬化、肌萎缩侧索硬化症 (ALS)、白血病和癌症等多种疾病，以及阿尔茨海默病和亨廷顿舞蹈病。而NPC的蛋白质成分和许多可溶性核运输因素已被确定并进行了广泛研究，双向运输的机制在不堵塞毛孔的情况下发生的情况仍然未知。 NPC具有近似八边形对称的形状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 在人类中。堵塞这个毛孔并装饰它的出口是一个由超过 200 个本质上无序的移动体组成的网络具有数千个苯丙氨酸-甘氨酸 (FG) 重复基序的多肽，为核提供结合位点通过 NPC 运送货物的转运受体 (NTR)。在稳定状态下，最多可达约 100 个 NTR 不对称地分布在整个 FG 网络中。异构动态 NTR/FG 网络建立渗透性屏障，同时为输入和输出的易位提供途径导出各种尺寸、亲和力和表面特性的复合物。多个首选路径存在中心孔。然而，这些的重叠、选择性以及几何和功能特性由于历史上缺乏剖析这些易位管道的技术工具，因此在很大程度上尚未探索过易位管道。具有必要的空间和时间分辨率的路径。为了解决这个缺陷，一种多色三色三维（3D）超分辨率荧光显微镜方法是在最后资助期间开发的能够结合单个功能 NPC 来确定单个功能 NPC 的位置和方向过境货物的粒子轨迹。该方法将用于确定结构和功能多个易位管道和 FG 网络屏障的特性。具体目标是： 1) 确定蛋白质易位导管的数量和性质； 2)确定FG-多肽和NTR FG 网络内的分布。目标 1 寻求探索至少三种不同易位的可能性导管，其中是否有 8 个不同的通道组成，以及是否有专用于任一进口或出口。目标 2 旨在确定 FG- 组件的物理排列和属性如何网络与促进已确定的易位管道相关联。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避免预期竞争和堵塞的方法用于通过同一通道的双向流量。此外，改进的超分辨率显微镜技术算法预计将构成该领域必要的工具包并具有广泛的适用性。