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

• 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
• 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

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.

项目总结/摘要 机械力在调节包括发育在内的许多生物过程中起着关键作用 和组织内环境稳定。在细胞水平，机械力的变化影响核功能。比如说， 细胞通过改变转录因子的定位，染色体组织， 和基因表达。尽管有这些见解，施加在原子核上的力是如何被解码的， 下游细胞反应仍然不清楚。核孔复合物（Nuclear Pore Complex，NPCs）是一种大量的蛋白质 通道，控制所有分子交换通过核膜（NE）。因此，NPC可以充当 通过对NE张力的变化做出功能性反应来翻译机械信号的中介。我 假设NE上张力改变NPC的构象，从而影响NPC的中枢转运 以影响其传输特性的方式传输。在这里，我将在目标1中直接探讨这一假设 通过使用两种互补的方法测试NPC如何响应NE张力的变化， 断层扫描（ET）和荧光共振能量转移（FRET）之间的FRET对工程化到 NPC支架。虽然ET将用于检查核孔径的变化，FRET将用于检查核孔径的变化。 在不同的张力状态下，使用遗传和 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， 光漂白（FRAP））的一系列荧光蛋白的报告，我可以在变化的背景下， 目的1中观察到的NPC构象。这些体内实验将与体外实验一起进行。 一种使用光镊的方法来观察NPC上的扩散如何响应NE的直接调制 纯化核上的张力。这些目标的完成将确定东北张力如何影响结构， NPCs的功能，并在细胞中建立新的机械传感通路。