Nuclear speckle liquid-liquid phase separation dynamics in senescence and aging

摘要 有机体的健康需要一个一致和平衡的内部环境，称为稳态。不同 生理过程在细胞水平上维持生物分子的适当水平，其中一些生物分子在细胞水平上维持适当水平。 机制随着年龄的增长而失效。蛋白质稳态，维持内质网中正确折叠的蛋白质水平 内质网（ER）是由未折叠蛋白反应（UPR）维持的。ER中过度错误折叠的蛋白质 激活UPR的三个分支，促进适应性过程，以恢复平衡的蛋白质组， cell.衰老与蛋白质稳态的丧失和衰老细胞的积累有关， 更长的复制和分泌促炎信号-表现出功能失调的UPR。分子 衰老细胞中UPR改变的潜在机制尚不清楚。我们假设液-液 核生物分子凝聚物的相分离（LLPS）动力学，核斑点（NS）， 衰老到普遍定期审议。独立于24小时时钟运行的12小时、依赖于XBP 1的时钟 或者细胞周期建立NS LLPS动力学的12小时超日节律。这些节律调节NS 形态和流动性通过SON，NS核心蛋白。高SON水平产生弥漫性、流体性NS和增强 UPR相关基因的表达。相比之下，低SON水平导致球形、停滞的NS和球形、停滞的NS。 UPR基因的表达减弱。我们最近发现SON水平降低，NS变得 在细胞衰老时更呈球形。这些数据表明，NS LLPS动力学的变化是 细胞衰老的标志在这里，我们提出了两个目的，以研究如何NS LLPS动态 在体外细胞衰老过程中和在体内整个时间老化过程中的变化。在第一个目标中，我们将 使用具有GFP标记的NS的小鼠胚胎成纤维细胞系，其可以被诱导进入衰老。这 模型将研究在衰老过程中NS LLPS动力学的12小时节律如何变化，以及如何 恢复SON水平影响衰老细胞中的NS LLPS动力学。我们还将大力推动 NS的扩散，以确定其流动性是否可以在衰老过程中增加。第二个目标将使用一个 秀丽隐杆线虫（C. habditis elegans）elegans）模型。我们将研究如何NS LLPS动力学 在整个衰老过程中发生变化，如果遗传和药理学方法使NS在 哺乳动物细胞可以类似地影响C. elegans和增强老年生物体中的蛋白质稳态。 这些目标将确立NS LLPS动力学的变化作为衰老和老化的标志。此外，委员会认为， 我们打算证明NS LLPS动力学是一个药物靶点，治疗可以使NS 形态和流动性达到衰老前状态。

Abstract Organismal health requires a consistent and balanced internal environment known as homeostasis. Different physiological processes maintain proper levels of biomolecules at a cellular level, and several of these mechanisms lose efficacy with age. Proteostasis, sustained levels of correctly folded proteins in the endoplasmic reticulum (ER), is maintained by the Unfolded Protein Response (UPR). Excessive misfolded proteins in the ER activate the three branches of the UPR, facilitating adaptive processes to restore a balanced proteome in the cell. Aging is associated with the loss of proteostasis and the accumulation of senescent cells – cells that no longer replicate and secrete pro-inflammatory signals – that exhibit a dysfunctional UPR. The molecular mechanisms underlying the altered UPR in senescent cells are unclear. We hypothesize that the liquid-liquid phase separation (LLPS) dynamics of a nuclear biomolecular condensate, the nuclear speckle (NS), link cellular senescence to the UPR. The 12-hour, XBP1s-dependent clock that functions independently of the 24-hour clock or the cell cycle establishes 12-hour ultradian rhythms of NS LLPS dynamics. These rhythms regulate NS morphology and fluidity through SON, the NS core protein. High SON levels create a diffuse, fluid NS and boost the expression of UPR-associated genes. In contrast, low SON levels result in a spherical, stagnant NS and a blunted expression of UPR genes. We have recently found that SON levels decrease, and that the NS becomes more spherical during cellular senescence. These data suggest that changes to NS LLPS dynamics are hallmarks of cellular senescence and aging. Here, we propose two aims to examine how NS LLPS dynamics change in vitro during cellular senescence and in vivo throughout chronological aging. In the first aim, we will use a mouse embryonic fibroblast line with a GFP-tagged NS that can be induced to enter senescence. This model will examine how established 12-hour rhythms of NS LLPS dynamics change during senescence and how restoring SON levels affects NS LLPS dynamics in senescent cells. We will also pharmacologically boost the diffuseness of the NS to determine if its fluidity can be increased during senescence. The second aim will use a Caenorhabditis elegans (C. elegans) model with a GFP-tagged NS. We will examine how NS LLPS dynamics change throughout aging and if genetic and pharmacological methods that make the NS more diffuse in mammalian cells can similarly affect NS LLPS dynamics in C. elegans and boost proteostasis in aged organisms. These aims will establish changes to NS LLPS dynamics as hallmarks of senescence and aging. Furthermore, we intend to show that NS LLPS dynamics is a druggable target and that therapies could return the NS morphology and fluidity to a pre-senescent state.