Molecular analysis of nuclear lamin assembly

核纤层组装的分子分析

• 批准号: 10231281
• 负责人: Ross T Pedersen
• 金额: $ 6.6万
• 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/10231281
• 关键词: Actins; Analytical Biochemistry; Behavior; Binding; Binding Proteins; Biochemical; Biological Assay; Caliber; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Cellular biology; Chromatin; Chromosomes; Complex; Crude Extracts; Cytoplasm; Cytoprotection; DNA; Data; Disease; ES Cell Line; Electron Microscopy; Embryology; Excision; Filament; Fluorescence Microscopy; Foundations; Gene Expression; Genes; Genome; Geometry; Goals; Human; Image; Importins; In Vitro; Institution; Intermediate Filament Proteins; Intermediate Filaments; Interphase; Interphase Cell; Investigation; Knock-out; Knowledge; Laboratories; Lamins; Light; 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; 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; reconstitution; scaffold; stem cells

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 层层丝是后生核核的中心结构组织者。它们有助于核功能 通过控制核结构，将核等离子体与细胞质分开并将基因组组织到 不同监管的子域。许多疾病与层固定失调和异常有关 核结构，强调了这些分子的重要性。尽管lamins在正常状态下很重要 核功能，控制层固定组件的分子机制知之甚少。先前的研究 试图使用在变性条件下纯化的重组层粘连蛋白剖析层固定组件 通过去除变性剂，类似地重折叠并组装成丝状结构。现在很清楚 这些实验中组装的层固定结构与细胞中的层粘膜丝不同。目标 该建议是开发用于研究物理层粘连蛋白组装的实验系统，并确定 层固定组件的组装途径和机制。期望这项研究扩展对 核结构和功能，以及与核结构和功能失调相关的疾病。 提出的实验旨在发现层粘连蛋白组装的分子机制。体外 实验将在包含自己的固体层层蛋白蛋白的爪蟾laevis卵提取物中进行 消除了组装测定中重组层粘连的需求。爪蟾鸡蛋提取物可以组装多样 层粘连结构。通过改变装配条件并使用荧光和 将确定控制层固定组件的电子显微镜，细胞结构和信号。使用分析 生物化学，可溶性层lamin亚基将被表征，以及稳定复合物中的任何蛋白质 与可溶性层固定亚基。 Importin和是可溶性层粘连蛋白的已知结合伴侣 提炼。提出的实验将确定进口蛋白和其他层粘连蛋白结合蛋白如何调节层粘连蛋白 集会。体内实验将在基因组编辑的干细胞中进行。小鼠胚胎干细胞与 编码所有三种层粘连同工型的基因已被宿主实验室分离并传播。 预计这些细胞中诱导表达荧光标记的层蛋白会导致新生的层粘连蛋白网格 组装，允许使用荧光和电子的层粘连组件成功可视化 显微镜。通过比较荧光标记的层固定突变体与野生型层状的组装的组装， 研究将确定层粘连组件途径是否因引起疾病的层固定突变而改变。 提出的研究将在卡内基机构的Yixian Zheng博士实验室进行 科学胚胎学系。研究将独立进行双周指导 由Zheng博士提供。实验培训以及科学写作和演讲培训将是 通过Zheng博士和实习生之间的一对一互动以及参与 卡内基机构的合作，合作和互动环境。