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

项目摘要/摘要 自噬是一种保护性的细胞机制，具有维持细胞器动态平衡的能力，从而 延缓细胞和组织的衰老。有趣的是，最近的研究表明， 细胞核对于维持时序寿命(CLS)可能很重要。与此一致的是，有许多 随着年龄的增长，细胞核和其结合膜成分之间长期存在的遗传联系。 因此，该领域的一个优先事项是充分了解核自噬(核吞噬)机制，以获得更多 充分界定清除核成分如何导致衰老和与衰老有关的疾病。然而， 在哺乳动物中没有明确的核货物适配器，因此将吞核作用与 自噬更具挑战性。相比之下，在萌芽中已经发现了一个亲核货物接头Atg39。 酵母菌提供了一个机会来研究吞核机制及其在维持CLS中的作用 这是一种模式生物。关键问题包括亲核需要哪些蛋白质，以及核是如何 包膜(NE)被重塑，以生成能够通过以下方式捕获的核的亚域 自噬小体。这项提案目标是定义嗜核行为的关键机制步骤，并提供 洞察其在延缓衰老方面的潜在作用。我将通过使用邻近标记和质量来实现这些目标 光谱分析以确定与Atg39合作驱动噬核的蛋白质。这些项目的贡献 还将通过测量目标基因缺失中的亲核通量来确定蛋白质对亲核的作用。 作为共定位实验，使用活细胞荧光显微镜，这将告知空间和 每种蛋白质功能的时间动态。第二个目标将利用创新的组合 双分子荧光互补(BIFC)和相关光电子显微镜(CLEM)研究 在超微结构分辨率上的亲核步骤。

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.