Modulation of nuclear pore complex structure and function by nuclear envelope tension

核膜张力调节核孔复杂结构和功能

基本信息

批准号：
10388820
负责人：
Megan R McCarthy
金额：
$ 5.49万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2023-03-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10388820
关键词：
Active Biological Transport Affect Architecture Binding Sites Biological Process Biophysics Caliber Cell Nucleus Cells Cellular biology Chromosome Structures Complex Cryo-electron tomography Cytoplasm Data Development Dextrans Diffusion Disease Engineering Extracellular Matrix Fission Yeast Fluorescence Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Gene Expression Genes Genetic Goals Homeostasis In Vitro Label Lead Malignant Neoplasms Measures Mechanics Mediating Modeling Molecular Molecular Conformation Monitor Nuclear Nuclear Envelope Nuclear Lamin Nuclear Pore Nuclear Pore Complex Nuclear Pore Complex Proteins Nucleoplasm Organism Outcome Pathway interactions Pharmacology Play Property Proteins Publishing Radial Reporter Role Sampling Scientist Series Signal Transduction Stretching Structure Testing Tissues Translating Yeasts base constriction electron tomography experimental study genetic manipulation in vivo insight laser tweezer macromolecule mechanical force mechanical signal mechanotransduction nucleocytoplasmic transport receptor response scaffold sensor temperature sensitive mutant tool transcription factor transmission process

项目摘要

Project Summary/Abstract Mechanical forces play a critical role in regulating numerous biological processes, including development and tissue homeostasis. At the cellular level, changes in mechanical forces impact nuclear function. For example, cells respond to mechanical inputs by altering the localization of transcription factors, chromosome organization, and gene expression. Despite these insights, how forces applied to the nucleus are decoded to affect downstream cellular responses remains poorly defined. Nuclear pore complexes (NPCs) are massive protein channels that control all molecular exchange across the nuclear envelope (NE). Thus, NPCs may serve as the intermediary that translates mechanical signals by functionally responding to changes in NE tension. I hypothesize that tension on the NE alters the conformation of NPCs, which affects the NPC central transport channel in a manner that influences its transport properties. Here, I will directly explore this hypothesis in Aim 1 by testing how the NPC responds to changes in NE tension using two complementary approaches, electron tomography (ET) and fluorescence resonance energy transfer (FRET) between FRET pairs engineered into the NPC scaffold. While ET will be used to examine changes in nuclear pore diameter, FRET will be used to examine dynamic changes in NPC structure in living cells under various tensional states induced using genetic and environmental perturbations. In a complementary but independent Aim 2, I will test how NE tension impacts the function of NPCs by interrogating the localization and dynamics (using fluorescence recovery after photobleaching (FRAP)) of a series of fluorescent protein reporters, which I can place in the context of changes in NPC conformation observed in Aim 1. These in vivo experiments will be performed alongside an in vitro approach using optical tweezers to observe how diffusion across the NPC responds to direct modulation of NE tension on purified nuclei. Completion of these Aims will determine how NE tension affects the structure and function of NPCs and establish a new mechanosensing pathway in cells.

项目概要/摘要机械力在调节包括发育在内的许多生物过程中发挥着关键作用和组织稳态。在细胞水平上，机械力的变化会影响核功能。例如，细胞通过改变转录因子、染色体组织的定位来响应机械输入，和基因表达。尽管有这些见解，施加到原子核的力如何被解码以影响下游细胞反应仍然不明确。核孔复合物 (NPC) 是大量蛋白质控制跨核膜 (NE) 的所有分子交换的通道。因此，NPC 可以作为通过对 NE 张力变化做出功能性响应来转换机械信号的中介体。我假设 NE 上的张力改变了 NPC 的构象，从而影响 NPC 中枢运输通道以影响其传输特性的方式。在这里，我将直接探讨目标1中的这个假设通过使用两种互补的方法（电子）测试 NPC 如何响应 NE 张力的变化断层扫描 (ET) 和 FRET 对之间的荧光共振能量转移 (FRET) NPC脚手架。 ET 将用于检查核孔径的变化，而 FRET 将用于检查利用遗传和诱导在各种张力状态下诱导活细胞中 NPC 结构的动态变化环境扰动。在补充但独立的目标 2 中，我将测试 NE 张力如何影响通过询问定位和动力学（使用荧光恢复后的 NPC 的功能）一系列荧光蛋白报告基因的光漂白（FRAP）），我可以将其置于变化的背景中在目标 1 中观察到的 NPC 构象中。这些体内实验将与体外实验一起进行使用光镊观察 NPC 扩散如何响应 NE 直接调制的方法纯化核上的张力。这些目标的完成将决定 NE 张力如何影响结构和 NPC 的功能并在细胞中建立新的机械传感途径。