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

摘要 有机体健康需要一个一致且平衡的内部环境，称为体内平衡。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 随着年龄的增长，机制会失去效力。蛋白质稳态，内质中正确折叠蛋白质的持续水平 网状结构 (ER) 由未折叠蛋白反应 (UPR) 维持。内质网中错误折叠蛋白过多 激活 UPR 的三个分支，促进适应性过程恢复平衡的蛋白质组 细胞。衰老与蛋白质稳态的丧失和衰老细胞的积累有关，这些细胞不 更长的时间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 改变的机制尚不清楚。我们假设液-液 核生物分子凝聚体的相分离 (LLPS) 动力学、核散斑 (NS)、链接细胞 普遍定期审议的衰老。依赖于 XBP1s 的 12 小时时钟，独立于 24 小时时钟运行 或者细胞周期建立 NS LLPS 动力学的 12 小时超昼夜节律。这些节律调节 NS 通过 SON（NS 核心蛋白）实现形态和流动性。高 SON 水平可产生弥散、流动的 NS 和增强效果 UPR相关基因的表达。相反，低 SON 水平会导致球形、停滞的 NS 和 UPR 基因的表达减弱。我们最近发现 SON 水平下降，并且 NS 变得 细胞衰老期间更呈球形。这些数据表明 NS LLPS 动态的变化 细胞衰老和衰老的标志。在这里，我们提出两个目标来研究 NS LLPS 动力学如何 在细胞衰老过程中的体外变化以及在整个时间老化过程中的体内变化。在第一个目标中，我们将 使用带有 GFP 标记 NS 的小鼠胚胎成纤维细胞系，可诱导其进入衰老状态。这个 该模型将研究衰老过程中 NS LLPS 动力学的已建立 12 小时节律如何变化以及如何变化 恢复 SON 水平会影响衰老细胞中的 NS LLPS 动态。我们还将从药理上增强 NS 的弥散度，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 带有 GFP 标记 NS 的秀丽隐杆线虫 (C. elegans) 模型。我们将研究 NS LLPS 动力学如何 随着年龄的增长，遗传和药理学方法会发生变化，使 NS 在体内更加扩散。 哺乳动物细胞同样可以影响线虫中的 NS LLPS 动态，并促进衰老生物体的蛋白质稳态。 这些目标将把 NS LLPS 动力学的变化作为衰老和衰老的标志。此外， 我们打算证明 NS LLPS 动力学是一个可药物靶标，并且治疗可以恢复 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.