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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要核膜维持核质和细胞质之间的区室化。在人类细胞的“有丝分裂”过程中，这种区划消失了，而染色体却被分离了。尽管膜上有一个洞，裂殖酵母粟酒裂殖酵母的“闭合有丝分裂”仍保持区室化初步数据表明，在纺锤体极体挤压过程中产生的核膜。与开放有丝分裂中负责核膜重组的机器相同，ESCRT 在粟酒裂殖酵母的每个细胞周期中，机械装置也负责密封这个孔，重要的是，这是一种分子机制。任何物种的核膜上的 ESCRT 活性都缺乏简单、单一、有丝分裂特异性的孔。粟酒裂殖酵母核膜封闭了每个细胞周期，为破译这种难以捉摸的现象提供了完美的系统该项目旨在为学员提供实现其长期目标所需的技能。此外，该项目将解决以下基本问题：跨物种核细胞生物学，符合 NIGMS 使命宣言。该项目提案分为两个目标 1：确定装配顺序和副本数量。驱动主轴极体挤压部位核包膜密封的因素；目标 2：询问每个因素在维持扩散屏障和/或驱动膜密封方面的贡献目标 1 将利用粟酒裂殖酵母系统的强大遗传学来研究核膜的超微结构。产生表达荧光标记的包膜核封闭因子结构的菌株，包括保守的 Heh1/Lem2-Cmp7/CHMP7 复合物和其他 ESCRT 蛋白将用于活细胞显微镜检查。确定组装顺序（通过参考细胞周期标记）以及拷贝数（通过基于 DNA 折纸的定量显微镜）在核膜闭合过程中的密封因子目标 2 将重点关注密封因素在其中发挥的作用的功能剖析。在膜闭合之前维持核区室并驱动膜重塑以密封核膜，通过使用时间精确的降解子方法来完成。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，同时冷冻电子断层扫描将允许建立第一个 ESCRT 介导的核膜密封的体内模型。学员将沉浸在高度协作和支持的环境中，同时完成拟议的课程该项目本身建立在受训者的许多现有技能的基础上，但重点是他获得一项技能。赞助商/共同赞助商（Lusk/King）共同监督内部的科学多样化实验室。耶鲁大学大学环境中出色的细胞生物学系学员将拥有。充分获得量身定制的专家指导、科学合作、口头演讲的机会，正式的教学/指导经验、科学推广机会以及与同行的联系。