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

摘要 生物体的健康需要一致和平衡的内部环境，即所谓的动态平衡。不同 生理过程在细胞水平上维持适当的生物分子水平，其中几个 这些机制会随着年龄的增长而失效。蛋白稳定，内质中正确折叠的蛋白质的持续水平 网状结构(ER)由未折叠蛋白反应(UPR)维持。内质网中过多的错误折叠蛋白质 激活UPR的三个分支，促进适应过程，以恢复平衡的蛋白质组 手机。衰老与蛋白平衡的丧失和衰老细胞的积累有关--这些细胞 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改变的潜在机制尚不清楚。我们假设液体-液体 核生物分子凝聚体的相分离(LLP)动力学，核散斑(NS)，链接细胞 对普遍定期审议的衰老。独立于24小时时钟运行的12小时XBP1s时钟 或者，细胞周期建立了NS LLP动力学的12小时超传统节律。这些节律调节NS 通过NS核心蛋白SON的形态和流动性。高SON水平会产生弥漫、流动的NS和助推 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基因表达迟钝。我们最近发现，SON水平下降，NS变成 在细胞衰老过程中更呈球形。这些数据表明，NS LLP动态的变化是 细胞衰老和衰老的特征。在这里，我们提出了两个目标来研究NS LLP的动态 细胞衰老过程中的体外变化和体内在整个时间老化过程中的变化。在第一个目标中，我们将 使用带有GFP标记的NS的小鼠胚胎成纤维细胞系，可以诱导其进入衰老。这 模型将研究NS LLP动力学12小时节律在衰老过程中如何变化以及如何 恢复SON水平影响衰老细胞中NS LLPS的动态变化。我们还将从药理上促进 测定NS的扩散性，以确定其在衰老过程中的流动性是否可以增加。第二个目标将使用 带有GFP标记NS的秀丽线虫模型。我们将研究NS LLP如何动态 随着年龄的增长而变化，如果遗传和药物方法，使NS更弥漫在 哺乳动物细胞可以类似地影响线虫的NS LLPS动态，并促进老年生物体的蛋白稳定。 这些目标将把NS LLP动态的变化确立为衰老和衰老的标志。此外， 我们打算证明NS LLP的动力学是一个可用药的靶点，治疗可以使NS 形态和流动性恢复到衰老前的状态。

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.