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

项目摘要。尽管细胞骨架在历史上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期间显现，这部分是由于 在这种情况下细胞骨架的破坏。有趣的是，细胞骨架的破坏 在衰老生物学领域，尽管缺乏明确的 直接证据。缺乏这些基本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，用于肌肉，肠， 和C. elegans，利用LifeAct融合到荧光分子。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。 这项工作奠定了基础，我目前正在进行的工作，在确定新的调节肌动蛋白 细胞骨架在建立了一个系统来询问细胞骨架质量之后，我现在可以询问细胞中的新基因。 它们在肌动蛋白调节中的潜在作用。利用这个和其他平台，我进行了多方面的筛选， 方法，以确定新的肌动蛋白的遗传调节。这些研究结合了肌动蛋白的体内活细胞成像 丝，用已知的肌动蛋白细胞骨架调节剂进行合成致死性筛选， 分析和全基因组CRISPR-Cas9筛选经历肌动蛋白应激的生物体。相互参照 这些丰富的数据集揭示了基因的两个关键节点：1）染色质状态的修饰剂及其 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，我假设存在一个一般的染色质状态，以促进健康的转录组， 正常的细胞骨架形式和功能，这是随着年龄的增长而分解的。此外，健康的 代谢状态可以在染色质重塑的上游或独立于染色质重塑起作用， 细胞骨架健康在目标1.2中，我建议研究是否有任何已识别的过程可以在组织中发挥作用- 特异性方式和细胞非自主性方式，通过回答两个问题：1）是过表达的 染色质重塑或脂质稳态因子在单个组织中足以维持生物体寿命？ 以及2）这些基因在神经元中的过表达是否驱动外周血中肌动蛋白细胞骨架的保护， 纸巾？目的2使用两种生化方法评估细胞骨架功能。首先，邻近标记将 用于表征肌动蛋白的新型蛋白质相互作用物，这些相互作用物对于正确的形式和功能很重要。第二，我们 构建了一种生物化学方法的工具，用于以单细胞分辨率定量肌动蛋白功能。本研究将 打开了令人兴奋的研究途径，了解细胞骨架功能对生理衰老的作用。

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.