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.
Loss of nuclear-cytoplasmic compartmentalization has been associated with several pathological contexts including cancer cell migration, ageing, and neurodegeneration. This project aims to uncover fundamental mechanisms that maintain proper compartmentalization at the nuclear envelope across all species. New mechanistic insights from the proposed studies have the potential to inform treatments of disease.