Establishing a Mechanism for the Autophagic Degradation of Nuclear Components and its Relationship to Aging

点击翻译按钮获取中文摘要

Project Summary/Abstract Autophagy is a protective cellular mechanism with the capacity to maintain organelle homeostasis and thereby delay cellular and organismal aging. Interestingly, recent work suggests that degradation of components of the nucleus may be important for maintaining chronological lifespan (CLS). Consistent with this, there are many long-standing genetic links between the nucleus, and components of its bounding membranes, with aging. Thus, a priority for the field is to fully understand the nuclear autophagy (nucleophagy) mechanism to more fully define how clearance of nuclear components contributes to aging and age-related disease. However, there is no defined nuclear cargo adaptor in mammals making isolating the role of nucleophagy from autophagy more challenging. By contrast, a nucleophagy cargo adaptor, Atg39, has been identified in budding yeast providing an opportunity to investigate the nucleophagy mechanism and its role in maintaining CLS in this model organism. Key questions include which proteins are required for nucleophagy and how the nuclear envelope (NE) is remodeled to generate a subdomain of the nucleus competent for capture by autophagosomes. The goal of this proposal is to define key mechanistic steps in nucleophagy and provide insight into its potential role in slowing aging. I will achieve these goals by using proximity-labeling and Mass Spectrometry to identify proteins that cooperate with Atg39 to drive nucleophagy. The contribution of these proteins to nucleophagy will be determined by measuring nucleophagic flux in targeted gene deletions as well as colocalization experiments using live-cell fluorescence microscopy, which will inform the spatial and temporal dynamics of the function of each protein. The second aim will utilize an innovative combination of Bimolecular Fluorescence Complementation (BiFC) and Correlative Light Electron Microscopy (CLEM) to study the steps of nucleophagy at the resolution of the ultrastructure.