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

项目摘要。尽管细胞骨架历来被理解为 在细胞中，肌动蛋白的适当功能也是各种细胞通路所必需的。中国经济的崩溃 这些细胞过程在衰老和暴露在无数的压力下表现出来，这在一定程度上是由于 在这种情况下细胞骨架的分解。有趣的是，细胞骨架的分解 古往今来，衰老生物学领域的常识一直被采纳，尽管缺乏明确的 和直接证据。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体细胞成像。在我博士后生涯的早期，我 开发了一种强大的、特定于组织的活细胞成像系统，用于肌肉、肠道、 和线虫的皮下组织，利用LifeAct融合到荧光分子上。LifeAct-mRuby可靠地绑定到 F-肌动蛋白，使其表达的细胞中的功能性丝状肌动蛋白可视化。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描述。 这项工作为我目前正在进行的鉴定肌动蛋白的新调节子的工作奠定了基础 细胞骨架。在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 它们在肌动蛋白调控中的潜在作用。使用这个和其他平台，我进行了多管齐下的筛选 鉴定肌动蛋白新的基因调控因子的方法。这些研究结合了肌动蛋白的活体细胞成像 细丝，利用已知的肌动蛋白细胞骨架调节器进行合成致死性筛选，以及两者的转录组 经历肌动蛋白应激的生物体的分析和全基因组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)染色质状态的修饰物和它们的 下游转录调节基因和2)与脂质储存和全球脂质动态平衡有关的基因。 在目标1.1中，我假设存在一个普遍的染色质状态，以促进健康的转录组 适当的细胞骨架形态和功能，并随着年龄的增长而分解。此外，一个健康的 代谢状态可以作用于染色质重塑的上游，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，我建议研究是否有任何已识别的过程可以在组织中发挥作用- 具体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动态平衡因子是否足以维持生物寿命？ 2)这些基因在神经元中的过度表达是否驱动了外周肌动蛋白细胞骨架的保护 纸巾？目的2采用两种生化方法评估细胞骨架功能。首先，邻近标记将是 用于表征肌动蛋白的新的蛋白质相互作用因子，对于正确的形式和功能非常重要。第二，我们是 建立一种生化方法的工具，用单细胞分辨率量化肌动蛋白的功能。这项研究将 为了解细胞骨架功能在生理衰老中的作用开辟了令人兴奋的研究途径。

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.