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

项目总结/摘要 自噬是一种保护性细胞机制，具有维持细胞器稳态的能力， 延缓细胞和器官衰老。有趣的是，最近的研究表明， 细胞核可能对维持时序寿命（CLS）很重要。与此相一致的是， 细胞核及其边界膜的组成部分与衰老之间的长期遗传联系。 因此，该领域的一个优先事项是充分了解核自噬（nucleophagy）机制， 充分确定核成分的清除如何导致衰老和与年龄有关的疾病。然而，在这方面， 在哺乳动物中没有明确的核货物衔接子， 自噬更具挑战性。相比之下，已经在芽殖细胞中鉴定了一种噬核货物衔接子Atg 39， 酵母提供了一个机会，研究噬核机制及其在维持CLS中的作用， 这个模式生物关键的问题包括哪些蛋白质是噬核作用所必需的，以及细胞核是如何形成的。 包膜（NE）被重塑以产生核的亚结构域，该亚结构域能够被 自噬体该提案的目标是定义噬核作用中的关键机制步骤，并提供 深入了解其在减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 光谱法鉴定与Atg 39合作驱动噬核作用的蛋白质。这些人的贡献 通过测量靶基因缺失中的噬核通量， 作为共定位实验，使用活细胞荧光显微镜，这将告知空间和 每个蛋白质功能的时间动态。第二个目标将利用以下创新组合： 双分子荧光互补法（BiFC）和相关光电子显微镜（CLEM）研究 在超微结构的分辨率上的噬核步骤。

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.