Phase separation can be skin deep The skin's barrier arises from proliferative cells that generate a perpetual upward flux of terminally differentiating epidermal cells. Cells nearing the body surface suddenly lose their organelles, becoming dead cellular ghosts called squames. Working in mouse tissue, Garcia Quiroz et al. found that as differentiation-specific proteins accumulate in the keratinocytes, they undergo a vinegar-in-oil type of phase separation that crowds the cytoplasm with increasingly viscous protein droplets (see the Perspective by Rai and Pelkmans). Upon approaching the acidic skin surface, the environmentally sensitive liquid-like droplets respond and dissipate, driving squame formation. These dynamics come into play in human skin barrier diseases, where mutations cause maladapted liquid-phase transitions. Science, this issue p. eaax9554; see also p. 1193 Phase-separation sensors reveal abundant liquid-like organelles at the crux of skin barrier formation. INTRODUCTION Liquid-liquid phase separation of biopolymers has emerged as a driving force for assembling membraneless biomolecular condensates. Despite substantial progress, studying cellular phase separation remains challenging. We became intrigued by enigmatic, membraneless protein granules (keratohyalin granules, KGs) within the terminally differentiating cell layers of mammalian epidermis. As basal progenitors cease to proliferate and begin their upward journey toward the skin surface, they produce differentiation-specific proteins that accumulate within KGs. Upon approaching the surface layers, all cellular organelles and KGs are inexplicably lost, resulting in flattened, dead cellular ghosts (squames) that seal the skin as a tight barrier to the environment. RATIONALE In an unbiased proteome-wide in silico search for candidate phase-transition proteins, we previously identified a major KG constituent, filaggrin (FLG), whose truncating mutations are intriguingly linked to human skin barrier disorders. Using advanced tools to study phase-separation behavior in mammalian skin, we pursued the possibility that liquid-liquid phase separation might lie at the root of both epidermal differentiation and human disease. RESULTS We found that KGs are liquid-like condensates, which assemble as filaggrin proteins undergo liquid-liquid phase separation in the cytoplasm of epidermal keratinocytes. Disease-associated FLG mutations specifically perturbed or abolished the critical concentration for phase separation–driven assembly of KGs. By developing sensitive, innocuous phase-separation sensors that enable visualization and interrogation of endogenous liquid-liquid phase-separation processes in mice, we found that filaggrin’s phase-separation dynamics crowd the cytoplasm with increasingly viscous KGs that physically affect organelle integrity. Liquid-like coalescence of KGs was restricted by surrounding bundles of differentiation-specific keratin filaments. Probing deeper, we found that as epidermal cells approached the acidic skin surface, phase-transition proteins experienced a rapid, naturally occurring pH shift and dynamically responded, causing the dissipation of their liquid-like KGs to drive squame formation. CONCLUSION Through the biophysical lens of liquid-liquid phase separation, our findings shed fresh light on the enigmatic process of skin barrier formation. Our design and deployment of phase-separation sensors in skin suggest a general strategy to interrogate endogenous liquid-liquid phase separation dynamics across biological systems in a nondisruptive manner. Through engineering filaggrins, filaggrin disease–associated variants, and our phase-separation sensors, we unveiled KGs as abundant, liquid-like membraneless organelles. During terminal differentiation, filaggrin family proteins first fuel phase-separation–driven KG assembly and subsequently, KG disassembly. Their liquid-like and pH-sensitive properties ideally equip KGs to sense and respond to the natural environmental gradients that occur at the skin’s surface and to drive the adaptive process of barrier formation. Liquid-phase condensates have typically been viewed as reaction centers where select components (clients) become enriched for processing or storage within cells. Analogously, KGs may store clients, possibly proteolytic enzymes and nucleases, that are temporally released in a pH-dependent manner to contribute to the self-destructive phase of terminal differentiation. Additionally, however, we provide evidence for biophysical dynamics emerging from condensate assembly, as KGs interspersed by keratin filament bundles massively crowd the keratinocyte cytoplasm and physically distort adjacent organelles. This crowding precedes the ensuing environmental stimuli that trigger disassembly of KGs, enucleation, and possibly other cellular events linked to barrier formation. Overall, the dynamics of liquid-like KGs, actionable by the skin’s varied environmental exposures, expose the epidermis as a tissue driven by phase separation. Finally, we discovered that filaggrin-truncating mutations and loss of KGs are rooted in maladapted phase-separation dynamics, illuminating why associated skin barrier disorders are exacerbated by environmental extremes. These insights open the potential for targeting phase behavior to therapeutically treat disorders of the skin’s barrier. Environmentally regulated liquid-phase dynamics drive skin barrier formation. (A) Using phase-separation sensors, we show that as basal progenitors flux toward the skin surface, they display phase-separation–driven assembly of liquid-like droplets. (B) In late-granular cells, these droplets crowd the cytoplasm and dissolve as cells (1) undergo chromatin compaction. (C) Near the skin surface, a sudden shift in intracellular pH regulates liquid-phase dynamics to drive squame formation. At the body surface, skin’s stratified squamous epithelium is challenged by environmental extremes. The surface of the skin is composed of enucleated, flattened surface squames. They derive from underlying, transcriptionally active keratinocytes that display filaggrin-containing keratohyalin granules (KGs) whose function is unclear. Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. The dynamics of phase separation governed terminal differentiation and were disrupted by human skin barrier disease–associated mutations. We used fluorescent sensors to investigate endogenous phase behavior in mice. Phase transitions during epidermal stratification crowded cellular spaces with liquid-like KGs whose coalescence was restricted by keratin filament bundles. We imaged cells as they neared the skin surface and found that environmentally regulated KG phase dynamics drive squame formation. Thus, epidermal structure and function are driven by phase-separation dynamics.

中文翻译：

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液-液相分离驱动皮肤屏障形成

相分离可以是皮肤深层皮肤屏障源于增殖细胞，这些细胞产生终末分化表皮细胞的永久向上流动。靠近体表的细胞突然失去细胞器，变成死细胞鬼，称为鳞片。Garcia Quiroz 等人在小鼠组织中工作。发现随着分化特异性蛋白质在角质形成细胞中积累，它们会经历油包醋型相分离，使细胞质中的蛋白质液滴越来越粘稠（参见 Rai 和 Pelkmans 的观点）。接近酸性皮肤表面时，环境敏感的液体状液滴会发生反应并消散，从而形成鳞屑。这些动力学在人类皮肤屏障疾病中发挥作用，其中突变导致不适应的液相转变。科学，这个问题 eaax9554; 另见第 1193 相分离传感器在皮肤屏障形成的关键处揭示了丰富的类似液体的细胞器。引言 生物聚合物的液-液相分离已成为组装无膜生物分子缩合物的驱动力。尽管取得了重大进展，但研究细胞相分离仍然具有挑战性。我们对哺乳动物表皮终末分化细胞层中神秘的无膜蛋白颗粒（透明角质颗粒，KGs）产生了兴趣。随着基础祖细胞停止增殖并开始向皮肤表面上行，它们会产生在 KG 内积累的分化特异性蛋白质。接近表层时，所有细胞器和KGs都莫名其妙地丢失，导致扁平化，死细胞鬼（鳞片）将皮肤密封起来，作为与环境的紧密屏障。基本原理在对候选相变蛋白的无偏见全蛋白质组计算机计算机搜索中，我们之前确定了一种主要的 KG 成分，丝聚蛋白 (FLG)，其截断突变与人类皮肤屏障障碍密切相关。使用先进的工具研究哺乳动物皮肤中的相分离行为，我们探索了液-液相分离可能是表皮分化和人类疾病的根源的可能性。结果 我们发现 KGs 是液体样的凝聚物，随着丝聚蛋白在表皮角质形成细胞的细胞质中进行液-液相分离而组装。疾病相关的 FLG 突变特别扰乱或消除了相分离驱动的 KG 组装的临界浓度。通过开发灵敏、无害的相分离传感器，使小鼠的内源性液-液相分离过程可视化和询问，我们发现丝聚蛋白的相分离动力学使细胞质充满越来越粘稠的 KG，这些 KG 会物理影响细胞器的完整性。KGs 的液体样聚结受到周围分化特异性角蛋白丝束的限制。深入探究，我们发现当表皮细胞接近酸性皮肤表面时，相变蛋白经历了快速、自然发生的 pH 值变化并动态响应，导致其液体状 KG 的消散驱动鳞屑形成。结论 通过液-液相分离的生物物理学视角，我们的研究结果揭示了皮肤屏障形成的神秘过程。我们在皮肤中设计和部署相分离传感器提出了一种通用策略，以无中断的方式询问生物系统中的内源性液-液相分离动力学。通过工程聚丝蛋白、聚丝蛋白疾病相关变体和我们的相分离传感器，我们揭示了 KG 作为丰富的液体状无膜细胞器。在终末分化过程中，聚丝蛋白家族蛋白首先促进相分离驱动的 KG 组装，随后促进 KG 分解。它们的液体状和 pH 敏感特性理想地使 KG 能够感知和响应发生在皮肤表面的自然环境梯度，并推动屏障形成的适应性过程。液相冷凝物通常被视为反应中心，其中选择的成分（客户）变得富集以在细胞内进行处理或储存。类似地，KG 可以储存客户，可能是蛋白水解酶和核酸酶，它们以 pH 依赖性方式暂时释放，以促进终末分化的自毁阶段。然而，此外，我们为凝聚物组装产生的生物物理动力学提供了证据，因为被角蛋白丝束散布的 KG 大量挤满了角质形成细胞的细胞质，并在物理上扭曲了相邻的细胞器。这种拥挤先于随后的环境刺激，这些刺激触发了 KGs 的分解、去核以及可能与屏障形成相关的其他细胞事件。总体而言，液体状 KGs 的动力学，可通过皮肤不同的环境暴露来操作，将表皮暴露为由相分离驱动的组织。最后，我们发现丝聚蛋白截断突变和 KGs 的丢失源于不适应的相分离动力学，阐明了为什么相关的皮肤屏障疾病会因极端环境而加剧。这些见解开启了靶向相位行为以治疗皮肤屏障疾病的潜力。环境调节的液相动力学驱动皮肤屏障的形成。(A) 使用相分离传感器，我们表明随着基底祖细胞流向皮肤表面，它们显示了相分离驱动的类液体液滴组装。(B) 在晚期颗粒细胞中，这些液滴聚集在细胞质中并随着细胞 (1) 进行染色质压实而溶解。(C) 在皮肤表面附近，细胞内 pH 值的突然变化调节液相动力学以驱动鳞屑形成。在体表，皮肤的分层鳞状上皮受到极端环境的挑战。皮肤表面由去核、扁平的表面鳞屑组成。它们源自潜在的、具有转录活性的角质形成细胞，这些角质形成细胞显示含有丝聚蛋白的透明角质颗粒 (KG)，其功能尚不清楚。在这里，我们发现丝聚蛋白通过液-液相分离组装 KG。相分离的动力学控制着终末分化，并被人类皮肤屏障疾病相关的突变所破坏。我们使用荧光传感器来研究小鼠的内源性相行为。表皮分层过程中的相变使细胞空间充满液体状 KG，其聚结受到角蛋白丝束的限制。我们对接近皮肤表面的细胞进行成像，发现环境调节的 KG 相动力学驱动鳞屑形成。因此，表皮结构和功能是由相分离动力学驱动的。我们对接近皮肤表面的细胞进行成像，发现环境调节的 KG 相动力学驱动鳞屑形成。因此，表皮结构和功能是由相分离动力学驱动的。我们对接近皮肤表面的细胞进行成像，发现环境调节的 KG 相动力学驱动鳞屑形成。因此，表皮结构和功能是由相分离动力学驱动的。