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

项目摘要/摘要 核膜维持核质和细胞质之间的区隔。在“开放”中 人类细胞的“有丝分裂”，当染色体被分离时，这种区划消失了。矛盾的是， 裂解酵母S.pombe的“闭合有丝分裂”尽管细胞膜上有一个洞，但仍保持区域化。 在主轴极体挤压过程中产生的核包膜。初步数据显示， 在开放有丝分裂中负责核膜重组的相同机械，ESCRT 机械，也负责封闭这个洞在S.pombe的每个细胞周期。关键的是，一种分子机制 在任何物种的核膜上都缺乏ESCRT活性。简单的、单一的、有丝分裂特有的孔 封闭了每个细胞周期的S.pombe核膜提供了完美的系统来破译这一难以捉摸的现象。 机制。该项目旨在为学员提供实现其长期目标所需的技能。 领导一个独立的研究小组。此外，该项目将涉及以下基本方面 跨物种的核细胞生物学，与NIGMS的使命声明一致。 该项目提案分为两个目标。目标1：确定《纽约时报》的组装顺序和拷贝数量 驱动主轴极体挤压部位核包膜密封的因素；目标2：询问 在以下情况下，维持扩散屏障和/或驱动膜密封的每个因素的贡献 核膜的超微结构。目标1将利用S.pombe系统的强大遗传学来 产生表达荧光标记的核膜封闭因子构建体的菌株，包括 保守的Heh1/Lem2-Cmp7/CHMP7复合体和其他ESCRT蛋白。活细胞显微镜将被用于 确定组装的顺序(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)在关闭核膜过程中的密封因子 主轴杆体挤压。目标2将侧重于对密封因子在其中扮演的角色的功能剖析 在膜关闭前维持核室，并驱动膜重塑以密封 核包层，通过使用时间上精确的简并方法来完成。相关的光和 电子显微镜将被用来严格评估每种蛋白质在空洞闭合中的作用，而冷冻- 电子断层扫描将为ESCRT介导的核膜封闭提供第一个活体模型。 学员将沉浸在高度协作和支持的环境中，同时完成建议的 项目。该项目本身建立在学员的许多现有技能的基础上，但重点是他获得 技术种类繁多。赞助商/联合赞助商(Lusk/King)共同监督一个科学多样化的实验室 耶鲁大学大学环境中的现象级细胞生物学系。实习生将拥有 充分获得量身定做的专家指导、科学合作、口头陈述的机会， 正式的教学/指导经验、科学外展机会以及与同龄人建立联系。

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.