Molecular analysis of nuclear lamin assembly

核纤层组装的分子分析

• 批准号: 10589892
• 负责人: Ross T Pedersen
• 金额: $ 7.18万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10589892
• 关键词: Actins; Analytical Biochemistry; Behavior; Binding; Binding Proteins; Biochemical; Biological Assay; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Cellular biology; Chromatin; Chromosomes; Complex; Crude Extracts; Cytoplasm; Cytoprotection; DNA; Data; Diameter; Disease; ES Cell Line; Electron Microscopy; Embryology; Excision; Filament; Fluorescence Microscopy; Foundations; Gene Expression; Genes; Genetic Transcription; Genome; Geometry; Goals; Human; Image; Importins; In Vitro; Institution; Intermediate Filament Proteins; Intermediate Filaments; Interphase; Interphase Cell; Investigation; Knock-out; Knowledge; Laboratories; Lamins; Measures; Mechanics; Microfilaments; Microtubules; Modeling; Molecular; Molecular Analysis; Monomeric GTP-Binding Proteins; Mus; Mutant Strains Mice; Mutation; Nuclear; Nuclear Envelope; Nuclear Lamin; Nuclear Lamina; Nuclear Pore Complex; Nuclear Structure; Nucleoplasm; Pathway interactions; Physiological; Play; Polymers; Property; Protein Isoforms; Proteins; Reaction; Recombinants; Regulation; Research; Role; Running; Science; Signal Transduction; Source; Structure; System; Testing; Training; Visualization; Work; Writing; Xenopus; Xenopus laevis; collaborative environment; disease-causing mutation; egg; embryonic stem cell; established cell line; experimental study; genome editing; in vitro Assay; in vivo; insight; mutant; novel; null mutation; physical insult; protein purification; reconstitution; scaffold; stem cells; success

Project Summary Lamin filaments are central structural organizers of the metazoan nucleus. They contribute to nuclear function by controlling nuclear structure, separating the nucleoplasm from the cytoplasm and organizing the genome into differentially regulated subdomains. Many diseases are associated with lamin dysregulation and abnormal nuclear structure, underscoring the importance of these molecules. Despite the importance of lamins in normal nuclear function, molecular mechanisms controlling lamin assembly are poorly understood. Previous studies attempted to dissect lamin assembly using recombinant lamin proteins purified under denaturing conditions and simultaneously refolded and assembled into filamentous structures through removal of denaturant. It is now clear that the lamin structures assembled in these experiments do not resemble lamin filaments in cells. The goal of this proposal is to develop experimental systems for studying physiological lamin assembly and to determine the assembly pathway and mechanism of lamin assembly. The research is expected to extend understanding of nuclear structure and function, and of diseases associated with dysregulation of nuclear structure and function. The proposed experiments aim to uncover molecular mechanisms of lamin assembly. In vitro experiments will be conducted in Xenopus laevis egg extracts, which contain their own soluble lamin protein, eliminating the need for recombinant lamins in assembly assays. Xenopus egg extracts can assemble diverse lamin structures. By varying assembly conditions and studying these lamin assemblies using fluorescence and electron microscopy, cellular structures and signals that control lamin assembly will be identified. Using analytical biochemistry, the soluble lamin subunit will be characterized, along with any proteins that are in a stable complex with the soluble lamin subunit. Importin  and  are known binding partners of soluble lamin in Xenopus egg extract. Proposed experiments will determine how importins and other lamin-binding proteins regulate lamin assembly. In vivo experiments will be conducted in genome edited stem cells. Mouse embryonic stem cells with the genes encoding all three lamin isoforms knocked out have been isolated and propagated by the host lab. Inducibly expressing fluorescently tagged lamin in these cells is predicted to result in nascent lamin meshwork assembly, allowing visualization of the succession of lamin assembly using fluorescence and electron microscopy. By comparing assembly of fluorescently tagged lamin mutants to assembly of wild type lamins, the research will determine whether the lamin assembly pathway is altered by disease-causing lamin mutations. The research proposed will be conducted in the laboratory of Dr. Yixian Zheng at the Carnegie Institution for Science Department of Embryology. Research will be carried out independently with biweekly guidance provided by Dr. Zheng. Experimental training, along with training in science writing and presentation, will be accomplished through one-on-one interactions between Dr. Zheng and the trainee and through participation in the collegial, collaborative, and interactive environment of the Carnegie Institution.

项目摘要 核纤层丝是后生动物细胞核的中心结构组织者。它们有助于核功能 通过控制核结构，将核质从细胞质中分离，并将基因组组织成 差异调节的亚结构域。许多疾病与核纤层蛋白的失调和异常 核结构，强调了这些分子的重要性。尽管核纤层蛋白在正常 核功能，控制核纤层蛋白组装的分子机制知之甚少。以前的研究 试图使用在变性条件下纯化的重组核纤层蛋白来剖析核纤层蛋白装配， 同时重折叠并通过去除变性剂组装成丝状结构。现在很清楚 在这些实验中组装的核纤层蛋白结构不类似于细胞中的核纤层蛋白丝。的目标 该建议是开发用于研究生理核纤层蛋白组装的实验系统， 核纤层蛋白的组装途径和组装机制。这项研究预计将扩大人们对 核结构和功能，以及与核结构和功能失调相关的疾病。 实验旨在揭示核纤层蛋白组装的分子机制。体外 实验将在非洲爪蟾卵提取物中进行，该卵提取物含有它们自身的可溶性核纤层蛋白， 消除了在组装测定中对重组核纤层蛋白的需要。非洲爪蟾卵提取物可以组装多种 核纤层结构通过改变组装条件，并使用荧光和免疫荧光技术研究这些核纤层蛋白组装体， 电子显微镜、细胞结构和控制核纤层蛋白组装的信号将被鉴定。使用分析 在生物化学中，可溶性核纤层蛋白亚基将与任何处于稳定复合物中的蛋白质沿着被表征 可溶性核纤层蛋白亚基。Importin α和β是已知的爪蟾卵中可溶性核纤层蛋白的结合伴侣 提取物拟议的实验将确定如何importins和其他核纤层蛋白结合蛋白调节核纤层蛋白 组装件.体内实验将在基因组编辑的干细胞中进行。小鼠胚胎干细胞 编码敲除的所有三种核纤层蛋白同种型的基因已被宿主实验室分离和繁殖。 预测在这些细胞中诱导表达荧光标记的核纤层蛋白会导致新生核纤层蛋白网络 组装，允许使用荧光和电子显微镜观察核纤层蛋白组装的连续性。 显微镜通过比较荧光标记的核纤层蛋白突变体的组装与野生型核纤层蛋白的组装， 研究将确定核纤层蛋白装配途径是否被致病核纤层蛋白突变改变。 拟议的研究将在卡内基研究所郑益贤博士的实验室进行 胚胎学科学部的研究将独立进行，每两周进行一次指导 由郑博士提供。实验培训，沿着科学写作和演示培训，将 通过郑博士和学员之间的一对一互动以及参与 卡内基研究所的学院式、协作式和互动式环境。