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

项目总结/摘要 核膜维持核质和细胞质之间的区室化。在“开放 在人类细胞的“有丝分裂”中，这种区室化在染色体分离时丢失。奇怪的是， “封闭有丝分裂”的裂变酵母S。粟酒裂殖酵母尽管在膜上有一个洞， 在纺锤体极体挤压过程中产生的核被膜。初步数据显示， 在开放有丝分裂中，ESCRT是负责核膜重新组装的相同机制 在S.粟酒重要的是，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，有丝分裂特异性的孔， 色葡萄粟酒裂殖酵母核膜，密封每个细胞周期，提供了完美的系统来破译这个难以捉摸的 机制本项目旨在为受训者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，该项目还将解决以下基本问题： 跨物种的核细胞生物学，符合NIGMS使命声明。 该项目提案分为两个目标。目标1：确定组装顺序和拷贝数 在纺锤体杆体挤出部位驱动核膜密封的因素;目的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 核膜的超微结构。Aim 1将利用S.粟粒系统， 产生表达荧光标记的核膜密封因子构建体的菌株，包括 保守的Heh 1/Lem 2-Cmp 7/CHMP 7复合物和其它ESCRT蛋白。活细胞显微镜将用于 确定装配顺序（通过参考细胞周期标记）以及拷贝数（通过 DNA折纸为基础的定量显微镜）的封闭因子在关闭的核膜后， 主轴杆体挤压。目的2将集中在功能解剖的作用，密封因素发挥作用， 在膜闭合之前维持核隔室并驱动膜重塑以密封核隔室， 核包膜，通过使用时间精确的degron方法完成。相关的光和 电子显微镜将被用来严格评估每种蛋白质在孔闭合中的作用，而冷冻- 电子断层扫描将允许ESCRT介导的核膜密封的第一个体内模型。 受训者将沉浸在一个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受训者现有的许多技能之上，但重点是他获得一个 广泛的技术。申办者/共同申办者（Lusk/King）共同监督一个科学多样性实验室， 在耶鲁大学的学院环境中，受训者将有 充分获得量身定制的专家指导，科学合作，口头陈述的机会， 正式的教学/指导经验，科学推广机会，以及与同龄人的网络。

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.