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

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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.