More than just a load control: cytoskeletal form and function during aging

不仅仅是负荷控制：衰老过程中的细胞骨架形式和功能

• 批准号: 10622612
• 负责人: Ryo Higuchi-Sanabria
• 金额: $ 24.88万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10622612
• 关键词: Actin-Binding Protein; Actins; Adopted; Affect; Age; Aging; Autophagocytosis; Binding; Biochemical; Biology of Aging; Brothers; CRISPR screen; Caenorhabditis elegans; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Cytoskeleton; Data Set; Dedications; Deterioration; Endocytosis; Exhibits; Exocytosis; Exposure to; F-Actin; Foundations; G Actin; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Health; Heat-Shock Response; Homeostasis; Housekeeping Gene; Human; Intestines; Knowledge; Label; Lipids; Liquid substance; Longevity; Maintenance; Metabolic; Methods; Microfilaments; Molecular; Muscle; Nature; Neurons; Organelles; Organism; Pathway interactions; Peripheral; Physiological; Postdoctoral Fellow; Process; Protein Dynamics; Proteins; Protocols documentation; Proxy; Quality Control; Regulation; Research; Resolution; Role; Stress; Structure; Subcutaneous Tissue; System; Technology; Testing; Tissues; Tubulin; Ubiquitin; Visualization; Western Blotting; Work; biochemical tools; career; chromatin remodeling; experience; experimental study; fitness; gene network; human old age (65+); in vivo; live cell imaging; multicatalytic endopeptidase complex; novel; overexpression; preservation; prevent; programs; response; screening; tool; transcriptome; whole genome

Project Summary. Although the cytoskeleton has historically been understood as the structural framework of the cell, the proper function of actin is also required for a diverse array of cellular pathways. The collapse of these cellular processes manifests during aging and exposure to a myriad of stresses, which is in part due to the breakdown of the cytoskeleton under these conditions. Interestingly, the breakdown of the cytoskeleton throughout age has been adopted as common knowledge in the field of aging biology, despite the lack of clear and direct evidence. A major contributor to the lack of these essential studies is the lack of tools available for in vivo, live-cell imaging of the actin cytoskeleton in multi-cellular organisms. Early in my postdoctoral career, I developed a system for robust, tissue-specific, live-cell imaging of the cytoskeleton in the muscle, intestine, and hypodermis of C. elegans, utilizing LifeAct fused to a fluorescent molecule. LifeAct-mRuby reliably binds to F-actin, allowing visualization of functional, filamentous actin in the cells it is expressed. Using this system, I performed an exhaustive characterization of the decline of actin cytoskeletal integrity during aging. This work laid the foundation of my currently ongoing work in identification of novel regulators of the actin cytoskeleton. Having set up a system to interrogate cytoskeletal quality, I can now interrogate novel genes in their potential role for actin regulation. Using this and other platforms, I performed a multi-pronged screening approach to identify novel genetic regulators of actin. These studies combined in vivo live cell imaging of actin filaments, synthetic lethality screening with known regulators of the actin cytoskeleton, and both transcriptome analysis and whole genome CRISPR-Cas9 screening of organisms experiencing actin stress. Cross-referencing these rich datasets has revealed two critical nodes of genes: 1) modifiers of chromatin state and their downstream transcriptional regulators and 2) genes involved in lipid storage and global lipid homeostasis. In Aim 1.1, I hypothesize that a general chromatin state exists to promote a healthy transcriptome for proper cytoskeletal form and function, and that this breaks down as a function of age. Moreover, a healthy metabolic state can work either upstream of – or independent of – chromatin remodeling to also promote cytoskeletal health. In Aim 1.2, I propose to study whether any of the identified processes can function in a tissue- specific manner and a cell non-autonomous manner, by answering two questions: 1) is overexpression of chromatin remodeling or lipid homeostasis factors in a single tissue sufficient to preserve organismal lifespan? and 2) does overexpression of these genes in neurons drive protection of the actin cytoskeleton in peripheral tissue? Aim 2 uses 2 biochemical approaches to assess cytoskeletal function. First, proximity labeling will be used to characterize novel protein interactors of actin important for proper form and function. Second, we are building a tool for a biochemical approach for quantifying actin function with single cell resolution. This study will open exciting avenues of research in understanding the role of cytoskeletal function on physiological aging.

项目摘要。尽管细胞骨架历来被理解为细胞的结构框架 在细胞中，肌动蛋白的正常功能也是多种细胞途径所必需的。的崩溃 这些细胞过程在衰老和暴露于无数压力的过程中表现出来，部分原因是 在这些条件下细胞骨架的分解。有趣的是，细胞骨架的分解 尽管缺乏明确的认识，但整个年龄已被采纳为衰老生物学领域的常识。 和直接证据。缺乏这些重要研究的一个主要原因是缺乏可用于 多细胞生物体中肌动蛋白细胞骨架的体内活细胞成像。在我博士后生涯的早期，我 开发了一种系统，用于对肌肉、肠道、 和线虫的皮下组织，利用与荧光分子融合的 LifeAct。 LifeAct-mRuby 可靠地绑定到 F-肌动蛋白，允许在其表达的细胞中可视化功能性丝状肌动蛋白。使用这个系统，我 对衰老过程中肌动蛋白细胞骨架完整性的下降进行了详尽的表征。 这项工作为我目前正在进行的肌动蛋白新型调节剂的鉴定工作奠定了基础 细胞骨架。建立了一个询问细胞骨架质量的系统后，我现在可以询问新的基因 它们对肌动蛋白调节的潜在作用。利用这个平台和其他平台，我进行了多管齐下的筛选 识别肌动蛋白新型遗传调节因子的方法。这些研究结合了肌动蛋白的体内活细胞成像 丝，使用已知的肌动蛋白细胞骨架调节因子进行合成致死率筛选，以及转录组 对经历肌动蛋白应激的生物体进行分析和全基因组 CRISPR-Cas9 筛选。交叉引用 这些丰富的数据集揭示了基因的两个关键节点：1）染色质状态的修饰因子及其 下游转录调节因子和 2) 参与脂质储存和整体脂质稳态的基因。 在目标 1.1 中，我假设存在一般染色质状态以促进健康的转录组 适当的细胞骨架形式和功能，并且随着年龄的增长而分解。此外，一个健康的 代谢状态可以在染色质重塑的上游发挥作用，也可以独立于染色质重塑，从而促进 细胞骨架健康。在目标 1.2 中，我建议研究任何已识别的过程是否可以在组织中发挥作用—— 特定方式和细胞非自主方式，通过回答两个问题：1）是过度表达 单个组织中的染色质重塑或脂质稳态因素足以维持生物体的寿命？ 2）这些基因在神经元中的过度表达是否会驱动外周肌动蛋白细胞骨架的保护？ 组织？目标 2 使用 2 种生化方法来评估细胞骨架功能。首先，邻近标签将 用于表征肌动蛋白的新型蛋白质相互作用因子，对于正确的形式和功能很重要。其次，我们是 构建一种生化方法工具，以单细胞分辨率量化肌动蛋白功能。这项研究将 为理解细胞骨架功能对生理衰老的作用开辟了令人兴奋的研究途径。