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 拉明细丝是后生动物核的中心结构组织者。它们对核功能做出了贡献 通过控制核结构，将核质与细胞质分离，并将基因组组织成 差异化调控的亚域。许多疾病都与层蛋白失调和异常有关 原子核结构，强调这些分子的重要性。尽管椎板在正常情况下很重要 核功能，控制层蛋白组装的分子机制还知之甚少。以前的研究 尝试使用在变性条件下纯化的重组层粘连蛋白来剖析层粘连蛋白组装 通过去掉变性剂，同时折叠并组装成丝状结构。现在很清楚了 在这些实验中组装的层粘连结构与细胞中的层粘连蛋白细丝不同。的目标是 这项建议是开发研究生理性板层组装的实验系统，并确定 板层组装的组装途径和机制。这项研究有望扩大对 核结构和功能，以及与核结构和功能失调相关的疾病。 拟议的实验旨在揭示层粘连蛋白组装的分子机制。离体 实验将在非洲爪哇的卵提取液中进行，这种提取液含有自己的可溶性层蛋白， 在组装分析中不再需要重组层粘连蛋白。非洲爪哇的卵提取物可以组装成不同的 拉明结构。通过改变组装条件并使用荧光和电子显微镜研究这些层粘连组件 电子显微镜、细胞结构和控制板层组装的信号将被识别。使用分析 生物化学，将描述可溶性的层蛋白亚基，以及稳定的复合体中的任何蛋白质。 与可溶性层粘连蛋白亚基结合。Importin和是非洲爪蛙卵中可溶性Lamin的已知结合伙伴 提取。拟议的实验将确定重要蛋白和其他层粘连蛋白结合蛋白如何调控层粘连蛋白 集合。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 被敲除的所有三种层粘连蛋白亚型的编码基因已经被宿主实验室分离和繁殖。 在这些细胞中诱导表达荧光标记的层粘连蛋白被预测会导致新生的层粘连蛋白网络 组件，允许使用荧光和电子来可视化层层组件的连续 显微镜。通过比较荧光标记的Lamin突变体和野生型Lamin的组装， 研究将确定层粘连蛋白组装途径是否因致病的层粘连蛋白突变而改变。 拟议的研究将在卡内基研究所郑义贤博士的实验室进行 为胚胎学科学系工作。研究将独立进行，每两周指导一次 郑博士提供。实验培训，以及科学写作和演示的培训，将是 通过郑博士和实习生之间的一对一互动和参与 卡内基研究所的合作、协作和互动环境。