Liquid-liquid phase separation drives 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.