The paradox of 'closed mitosis': using fission yeast to decipher a molecular model of ESCRT activity at the nuclear envelope

“闭合有丝分裂”的悖论：使用裂殖酵母破译核膜上 ESCRT 活性的分子模型

• 批准号: 10294946
• 负责人: Nicholas Ryan Ader
• 金额: $ 6.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10294946
• 关键词: Ablation; Accounting; Address; Aging; Architecture; Automobile Driving; Biological Models; Carrier Proteins; Cell Cycle; Cells; Cellular biology; Characteristics; Chromosome Segregation; Chromosomes; Collaborations; Communities; Complex; Cryo-electron tomography; Cytoplasm; DNA; Data; Daughter; Diffusion; Disease; Dissection; Educational process of instructing; Electron Microscopy; Ensure; Environment; Eukaryota; Event; Family; Fibrinogen; Fission Yeast; Future; Genetic; Genetic Models; Genome; Goals; Human; In Situ; Lead; Light; Maintenance; Mechanics; Mediating; Membrane; Membrane Proteins; Mentors; Mentorship; Microscopy; Mission; Mitosis; Mitotic; Modeling; Molecular; Morphology; National Institute of General Medical Sciences; Nerve Degeneration; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nucleoplasm; Oral; Pathologic; Play; Polymers; Positioning Attribute; Process; Proteins; Quantitative Microscopy; Research; Role; Rupture; Saccharomycetales; Site; Sorting - Cell Movement; Supervision; System; Techniques; Testing; Training; Translating; Universities; base; cancer cell; cell motility; collaborative environment; college; design; emerin; experience; genetic manipulation; in vivo Model; innovation; insight; light microscopy; live cell microscopy; member; molecular modeling; nanometer; outreach; peer; recruit; repaired; seal; skills; spindle pole body; stoichiometry; success; supportive environment; tool

Project Summary/Abstract The nuclear envelope maintains compartmentalization between the nucleoplasm and the cytoplasm. In the “open mitosis” of human cells, this compartmentalization is lost while chromosomes are segregated. Paradoxically, the “closed mitosis” of the fission yeast S. pombe maintains compartmentalization despite a hole in the membranes of the nuclear envelope created during extrusion of the spindle pole body. Preliminary data suggest that the same machinery that is responsible for reassembly of the nuclear envelope in open mitosis, the ESCRT machinery, is also responsible for sealing this hole each cell cycle in S. pombe. Critically, a molecular mechanism of ESCRT activity at the nuclear envelope in any species is lacking. The simple, single, mitotic-specific hole in the S. pombe nuclear envelope, sealed every cell cycle, provides the perfect system to decipher this elusive mechanism. This project is designed to provide the trainee the skills necessary to reach his long-term goal to lead an independent research group. Additionally, the project will address fundamental aspects of nuclear cell biology across species, in line with the NIGMS Mission Statement. The project proposal is divided into two aims. Aim 1: Determine the order of assembly and copy number of the factors that drive nuclear envelope sealing at the site of spindle pole body extrusion; Aim 2: Interrogate the contribution of each factor in maintaining a diffusion barrier and/or driving membrane sealing in the context of the ultrastructure of the nuclear envelope. Aim 1 will leverage the powerful genetics of the S. pombe system to generate strains expressing fluorescently-tagged constructs of nuclear envelope sealing factors, including the conserved Heh1/Lem2-Cmp7/CHMP7 complex and other ESCRT proteins. Live-cell microscopy will be used to determine the order of assembly (through reference to cell cycle markers) as well as the copy number (through DNA origami-based quantitative microscopy) of sealing factors during closure of the nuclear envelope after spindle pole body extrusion. Aim 2 will focus on a functional dissection of the roles that sealing factors play in maintaining the nuclear compartment prior to membrane closure and driving membrane remodeling to seal the nuclear envelope, accomplished through the use of a temporally precise degron approach. Correlated light and electron microscopy will be employed to rigorously assess the role of each protein in hole closure, while cryo- electron tomography will allow for the first in vivo model for ESCRT-mediated nuclear envelope sealing. The trainee will be immersed in a highly collaborative and supportive environment while completing the proposed project. The project itself builds upon many of the trainee’s existing skills, but focuses on his acquisition of a broad array of techniques. The sponsor/co-sponsor (Lusk/King) co-supervise a scientifically diverse lab within the phenomenal Cell Biology department in the collegiate environment of Yale University. The trainee will have ample access to tailored expert mentorship, scientific collaborations, opportunities to give oral presentations, formal teaching/mentoring experiences, scientific outreach opportunities, and networking with peers.

项目摘要/摘要 核包膜维持核等离子体和细胞质之间的分区化。在“开放”中 人类细胞的有丝分裂，这种分区化丢失了，而染色体则被隔离。 裂变酵母S. pombe的“封闭有丝分裂”维持隔离目的地一个孔的孔 在纺锤体体延伸过程中产生的核包膜的。初步数据表明 相同的机械，负责在开放有丝分裂中重新组装核包膜， 机械还负责密封S. pombe中的每个细胞周期。至关重要的是分子机制 缺乏任何物种中核包膜的ESCRT活性。简单的，单一的有丝分裂特异性孔 密封每个单元格周期密封的S. pombe核包络，为破译这种难以捉摸的系统提供了完美的系统 机制。该项目旨在为学员提供实现长期目标所需的技能 领导一个独立的研究小组。此外，该项目将解决基本方面 跨物种的核细胞生物学与NIGMS任务陈述一致。 项目提案分为两个目标。目标1：确定组装和复制号的顺序 在纺锤体伸展部位驱动核包膜密封的因素；目标2：审问 在维持扩散屏障和/或驱动膜密封的情况下，每个因素的贡献 核包膜的超微结构。 AIM 1将利用S. Pombe系统的强大遗传学 产生表达荧光标记的核包膜密封因子的菌株，包括 保守的HEH1/LEM2-CMP7/CHMP7复合物和其他ESCRT蛋白。活细胞显微镜将用于 确定装配顺序（通过参考单元周期标记）以及拷贝数（通过 密封因子的基于DNA折纸的定量显微镜）在关闭核包络的密封因子之后 主轴杆身体延伸。 AIM 2将重点放在密封因子中扮演的角色的功能解剖上 在膜闭合之前保持核室并驱动膜重塑以密封 通过使用暂时精确的Degron方法来实现的核包络。相关的光和 电子显微镜将用于严格评估每种蛋白质在孔闭合中的作用，而冷冻 电子断层扫描将允许首个用于ESCRT介导的核包膜密封的体内模型。 在完成该提案的同时，学员将沉浸在高度协作和支持的环境中 项目。该项目本身以许多受训者的现有技能为基础，但专注于他对 广泛的技术。赞助商/共同发起人（Lusk/King）共同享受一个科学多样的实验室 耶鲁大学大学环境中的现象细胞生物学系。学员将有 可以充分访问量身定制的专家精力，科学合作，提供口头演示的机会， 正式的教学/指导经验，科学的外展机会以及与同龄人建立联系。