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Cracking the Skin Barrier: Liquid-Liquid Phase Separation Shines under the Skin

Open AccessPublished:July 05, 2021DOI:https://doi.org/10.1016/j.xjidi.2021.100036
      Central to forming and sustaining the skin’s barrier, epidermal keratinocytes (KCs) fluxing to the skin surface undergo a rapid and enigmatic transformation into flat, enucleated squames. At the crux of this transformation are intracellular keratohyalin granules (KGs) that suddenly disappear as terminally differentiating KCs transition to the cornified skin surface. Defects in KGs have long been linked to skin barrier disorders. Through the biophysical lens of liquid-liquid phase separation (LLPS), these enigmatic KGs recently emerged as liquid-like membraneless organelles whose assembly and subsequent pH-triggered disassembly drive squame formation. To stimulate future efforts toward cracking the complex process of skin barrier formation, in this review, we integrate the key concepts and foundational work spanning the fields of LLPS and epidermal biology. We review the current progress in the skin and discuss implications in the broader context of membraneless organelles across stratifying epithelia. The discovery of environmentally sensitive LLPS dynamics in the skin points to new avenues for dissecting the skin barrier and for addressing skin barrier disorders. We argue that skin and its appendages offer outstanding models to uncover LLPS-driven mechanisms in tissue biology.

      Abbreviations:

      3D (three-dimensional), AD (atopic dermatitis), CE (cornified envelope), EDC (epidermal differentiation complex), ER (endoplasmic reticulum), IDP (intrinsically-disordered protein), KC (keratinocyte), KG (keratohyalin granule), LCST (lower critical solution temperature), LLPS (liquid-liquid phase separation), PTM (post-translational modification), TG (trichohyalin granule), UCST (upper critical solution temperature)

      Toward cracking the skin barrier

      Skin is the largest and foremost defensive and sensory organ in the human body (
      • Pasparakis M.
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      Mechanisms regulating skin immunity and inflammation.
      ). At the interface with the external environment, the epidermal surface seals the skin as a tight and environmentally responsive tissue barrier. Essential to life, this mammalian skin barrier prevents water loss, excludes pathogens, and provides resistance to physical and chemical insults (
      • Madison K.C.
      Barrier function of the skin: "la raison d'etre" of the epidermis.
      ).
      The architecture and cellular dynamics of the epidermis are key to its protective barrier function. The epidermis is a stratified squamous epithelium in which transcriptionally active keratinocytes (KCs) constantly flux upward toward the skin surface. Self-renewal begins at the innermost basal layer of the epidermis, where epidermal stem cells divide and fuel terminal differentiation. Along their upward differentiation path, KCs acquire defining structural features demarcating three distinct stages: the spinous, granular, and corneum layers (
      • Fuchs E.
      Scratching the surface of skin development.
      ;
      • Moreci R.S.
      • Lechler T.
      Epidermal structure and differentiation.
      ). Under tissue homeostasis, mouse KCs flux from the basal to the granular layer with stereotypical transit times, but they proceed stochastically through the granular-to-corneum transition (
      • Rompolas P.
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      • Kawaguchi K.
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      Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.
      ). The granular layer appears to act as a buffer zone with yet unknown mechanisms to coordinate the formation of cornified tissue (
      • Rompolas P.
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      • Kawaguchi K.
      • Park S.
      • Gonzalez D.
      • Brown S.
      • et al.
      Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.
      ;
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      • Tanaka R.J.
      • Kajimura M.
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      Epidermal cell turnover across tight junctions based on Kelvin's tetrakaidecahedron cell shape.
      ). When KCs in the granular layer reside at the corneum interface, they abruptly transform into corneocytes by losing their nuclei and cytoplasmic organelles (
      • Eckhart L.
      • Lippens S.
      • Tschachler E.
      • Declercq W.
      Cell death by cornification.
      ). This KC-to-corneocyte transition is critical: corneocytes with squame features pile tightly, depositing and maturing intercorneocyte lipid structures that are key to the skin’s permeability barrier (
      • Narangifard A.
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      • den Hollander L.
      • Iwai I.
      • Han H.
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      ;
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      ). Because surface corneocytes are sloughed off in response to persistent environmental pressures, the underlying layers replenish the corneum to sustain barrier integrity (
      • Kubo A.
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      Epidermal barrier dysfunction and cutaneous sensitization in atopic diseases.
      ;
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      • Park S.
      • Gonzalez D.
      • Brown S.
      • et al.
      Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.
      ;
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      • Mertz A.F.
      • Kubo A.
      • Marg S.
      • Jüngst C.
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      ;
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      • Tanaka R.J.
      • Kajimura M.
      • Suematsu M.
      • et al.
      Epidermal cell turnover across tight junctions based on Kelvin's tetrakaidecahedron cell shape.
      ). This epidermal turnover in human skin is robust, estimated at about 3.7 billion cells per day—about 1% of the total daily cellular turnover in humans (
      • Sender R.
      • Milo R.
      The distribution of cellular turnover in the human body.
      ).
      The epidermal dynamics underlying cellular turnover in the skin are sensitive to environmental demands and shift dramatically on instances of barrier disruption and skin inflammation. Yet, the cellular mechanisms that couple epidermal differentiation and environmental pressures on the skin barrier remain largely unknown. Mounting evidence points to the granular layer as crucial to the formation of a functional and environmentally resilient cornified layer. For example, an immature or absent granular layer is strongly linked to human disorders involving abnormal cornification and dysfunctional skin barrier phenotypes exacerbated by environmental extremes (
      • Kantor R.
      • Silverberg J.I.
      Environmental risk factors and their role in the management of atopic dermatitis.
      ;
      • Mlitz V.
      • Latreille J.
      • Gardinier S.
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      • Drouault Y.
      • Hufnagl P.
      • et al.
      Impact of filaggrin mutations on Raman spectra and biophysical properties of the stratum corneum in mild to moderate atopic dermatitis.
      ;
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      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Smith F.J.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Sandilands A.
      • Campbell L.E.
      • Zhao Y.
      • et al.
      Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.
      ;
      • Thyssen J.P.
      • Jakasa I.
      • Riethmüller C.
      • Schön M.P.
      • Braun A.
      • Haftek M.
      • et al.
      Filaggrin expression and processing deficiencies impair corneocyte surface texture and stiffness in mice.
      ). These differentiation defects literally crack the barrier, offering mechanistic clues (
      • Martins A.F.
      • Bennett N.C.
      • Clavel S.
      • Groenewald H.
      • Hensman S.
      • Hoby S.
      • et al.
      Locally-curved geometry generates bending cracks in the African elephant skin.
      ) to figuratively crack the mechanisms underlying skin barrier formation.
      To unravel the epidermal dynamics and environmental responsiveness of the skin barrier in health and disease, we argue that the granular-to-corneum transition will require dissection at the tissue, cellular, and subcellular levels. Historically, tackling this challenge faced steep technical and conceptual barriers that prevented progress. Prominent among these barriers, we lacked biophysical tools and frameworks to predict, chart, and visualize the behavior of key proteins and cellular processes involved in this rapid and complex differentiation program. With recent progress toward addressing these gaps, as we will explain, the scientific landscape is ripe to crack enigmatic cellular mechanisms that underlie skin barrier formation.
      A subset of key proteins throughout epidermal differentiation are intrinsically-disordered proteins (IDPs) that defy the traditional interpretation of proteins as ordered molecular solids (Figure 1a). Long underappreciated in protein biology, IDPs recently emerged as major drivers of intracellular self-assembly. We and others have begun to chart their unique sequence-encoded properties in cells and across biological systems (
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ;
      • Wang J.
      • Choi J.M.
      • Holehouse A.S.
      • Lee H.O.
      • Zhang X.
      • Jahnel M.
      • et al.
      A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins.
      ), including skin (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Furthermore, epidermal cells that flux through the granular layer are typified by the emergence and loss of poorly understood IDP-rich, membraneless protein granules. These dynamic cytoplasmic structures, which have counterparts across cornifying epithelia, have been notoriously difficult to study. Excitingly, new tools and concepts from the multidisciplinary field of liquid-liquid phase separation (LLPS) now enable us to approach membraneless protein granules as functional and highly dynamic organelles (
      • Bergeron-Sandoval L.P.
      • Safaee N.
      • Michnick S.W.
      Mechanisms and consequences of macromolecular phase separation.
      ;
      • Shin Y.
      • Brangwynne C.P.
      Liquid phase condensation in cell physiology and disease.
      ). Building on these insights, we recently exposed keratohyalin granules (KGs), the protein granules of granular layer cells, as liquid-like membraneless organelles whose assembly and disassembly fuel skin barrier formation (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ).
      Figure thumbnail gr1
      Figure 1Phase separation-driven assembly of membraneless organelles. (a) IDPs exhibit dynamic structural fluctuations. In snapshots from four separate molecular dynamics simulations, a model IDP (left) fails to adopt a defined three-dimensional structure, whereas a helical protein domain (right) reproducibly folds into the same helical solid. Adapted from
      • Quiroz F.G.
      • Chilkoti A.
      The language of protein polymers.
      . (b) Resistant to folding, IDPs engage in extensive intermolecular interactions. When these sequence-dependent and stimuli-sensitive interactions increase over a critical threshold, an LLPS transition ensues: a large fraction of IDP molecules coalesce into condensates. As a model LLPS-IDP, we consider an IDP with a repetitive architecture. Repeats are not essential for LLPS but are common in IDPs and readily tune their LLPS behavior (
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ). (c) Membraneless organelles have properties akin to liquids such as (left to right) flow under shear force, rapid fusion between similar droplets, and selective fusion with dissimilar droplets. Intermolecular forces between scaffolds and/or LLPS-IDPs determine the viscosity and surface tension of these liquid-like organelles. Viscosity alters the speed of flow (gray arrows; left panel) and the kinetics of fusion events (middle panel). Less intuitively, the condensates assembled by distinct scaffolds or LLPS-IDPs are either miscible (top right panel) or immiscible (bottom right panel). The outcome of these organelle interactions ultimately depends on surface tension, surface charge, and the potential/affinity of cross-scaffold interactions (
      • Kaur T.
      • Raju M.
      • Alshareedah I.
      • Davis R.B.
      • Potoyan D.A.
      • Banerjee P.R.
      Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies.
      ). (d) Representative phase diagram as a function of environmental stimuli (in this situation, temperature) and protein concentration for polymers and IDPs that exhibit UCST LLPS. Above a critical point (the UCST), irrespective of LLPS-IDP concentration ([LLPS-IDP]), the system resides in the one-phase region (light gray): low intermolecular interactions between LLPS-IDP chains are never conducive to LLPS. To illustrate LLPS under the UCST, we consider a cell actively synthesizing an LLPS-IDP at a constant temperature. At t1, intracellular LLPS-IDP levels are below the Csat or critical concentration for phase separation—equivalent to CDilute under equilibrium. Under these conditions, LLPS-IDP molecules are diffuse and are well-mixed with other proteins in the cytoplasm (t1, bottom and right panels). On sustained protein synthesis, at t2, intracellular LLPS-IDP concentration increases over Csat, driving the system into the two-phase regime (dark gray). LLPS-IDP molecules now reside in one of the two phases: a condensate or high-density phase (at CDense) and a dilute phase (at CDilute). As a result, a fraction of LLPS-IDP molecules markedly demixed from other proteins in the cytoplasm (t2, bottom and right panels). Moving along the tie line (dashed line) as intracellular LLPS evolves from t2 to t3, further increases in total LLPS-IDP levels do not alter CDense or CDilute but increase the volume fraction of the condensate phase (fDense) at the expense of the dilute phase (fDilute). The progressive shift toward fDense > fDilute (purple gradients) involves increases in the number and/or size of LLPS condensates (t3, bottom and right panels). (e) PTMs may alter intermolecular interactions to either favor (left) or oppose (right) intracellular LLPS. (f) Model of multicomponent condensate assembly by multivalent scaffolds and low-valency clients. Heterotypic interactions between domains in multivalent scaffolds can lead to LLPS (
      • Li P.
      • Banjade S.
      • Cheng H.C.
      • Kim S.
      • Chen B.
      • Guo L.
      • et al.
      Phase transitions in the assembly of multivalent signalling proteins.
      ), even if the scaffold proteins are incapable of exhibiting LLPS on their own. These systems inevitably follow complex phase diagrams that are not captured by the single-component LLPS-IDP system in d. Clients are readily enriched in these LLPS condensates by binding to sites on the multivalent scaffold. The identity of these clients contributes to the varied functionality of membraneless organelles (
      • Banani S.F.
      • Rice A.M.
      • Peeples W.B.
      • Lin Y.
      • Jain S.
      • Parker R.
      • et al.
      Compositional control of phase-separated cellular bodies.
      ). Csat, saturation concentration; IDP, intrinsically-disordered protein; LLPS, liquid-liquid phase separation; LLPS-IDP, liquid-liquid phase separation‒exhibiting IDP; Nu, nucleus; PTM, post-translational modification; UCST, upper critical solution temperature.
      In this review, we integrate concepts, tools, and approaches spanning IDP research, LLPS, and skin biology as a step toward cracking the process of skin barrier formation. We expect that discussion of the biophysical frameworks and recent findings in the skin will equip biologists and clinicians with an LLPS-inspired lens to study the skin barrier. Using this lens, we illuminate new hypotheses at the interface of novel and known cellular mechanisms at play in barrier formation. As a corollary, for readers with a multidisciplinary background, this review exposes skin and its appendages as fascinating tissue and organ systems to examine LLPS-driven mechanisms.

      Phase separation-driven assembly of membraneless organelles

      How are cellular compartments that are not bound by lipid membranes, such as KGs in the skin, stabilized to function as organelles? How do they achieve selective component exchange with their surroundings? Mechanistically, how do their structural and molecular features contribute to cellular processes? Over the last decade, answers to these questions have emerged from the biophysical framework of LLPS (
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ;
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Fueled by thought-provoking observations of liquid-like behavior in germline-defining granules (
      • Brangwynne C.P.
      • Eckmann C.R.
      • Courson D.S.
      • Rybarska A.
      • Hoege C.
      • Gharakhani J.
      • et al.
      Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
      ), combined with progress on the sequence encoding of LLPS in IDPs (
      • Dzuricky M.
      • Roberts S.
      • Chilkoti A.
      Convergence of artificial protein polymers and intrinsically disordered proteins.
      ;
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ;
      • Wang J.
      • Choi J.M.
      • Holehouse A.S.
      • Lee H.O.
      • Zhang X.
      • Jahnel M.
      • et al.
      A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins.
      ), the stage is set to dissect the physiological roles of long-overlooked membraneless compartments in stratifying epithelia and across biological systems.
      Membraneless structures have long puzzled biologists. In the 1830s, the highly refractive nucleolus, visible through brightfield microscopy, emerged as the first documented intranuclear compartment (
      • Trinkle-Mulcahy L.
      Nucleolus: the consummate nuclear body.
      ). By the late 1890s, the use of fixatives and dyes exposed cytoplasmic granules linked to epidermal differentiation (
      • Holbrook K.A.
      Biologic structure and function: perspectives on morphologic approaches to the study of the granular layer keratinocyte.
      ) and to germline specification (
      • Hegner R.W.
      The germ cell determinants in the eggs of chrysomelid beetles.
      ,
      • Hegner R.W.
      Effects of removing the germ-cell determinants from the eggs of some chrysomelid beetles. Preliminary report.
      ). Additional biochemical methods revealed now-stereotypical membraneless organelles of the nucleus: Cajal bodies (
      • Cajal S.R.
      Un sencillo método de coloración selectiva del retículo protoplasmático y sus efectos en los diversos órganos nerviosos de vertebrados e invertebrados.
      ) and speckles (
      • Cajal S.R.
      El núcleo de las células piramidales del cerebro humano y de algunos mamíferos.
      ;
      • Gall J.G.
      The centennial of the Cajal body.
      ;
      • Lamond A.I.
      • Spector D.L.
      Nuclear speckles: a model for nuclear organelles.
      ). Crucially, beginning in the 1940s, high-resolution electron microscopy confirmed the membraneless nature of nucleoli (
      • Leblond C.P.
      The life history of cells in renewing systems.
      ;
      • Trinkle-Mulcahy L.
      Nucleolus: the consummate nuclear body.
      ) and of other intracellular granules, including KGs (
      • Lavker R.M.
      • Matoltsy A.G.
      Substructure of keratohyalin granules of the epidermis as revealed by high resolution electron microscopy.
      ) and germline-defining granules (
      • Mahowald A.P.
      Fine structure of pole cells and polar granules in Drosophila melanogaster.
      ). Later in the 1980s, benefiting from immunofluorescence,
      • Strome S.
      • Wood W.B.
      Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.
      documented the germline granules of Caenorhabditis Elegans, coining the term P granules. Despite this progress, imaging fixed cells reinforced the view of membraneless structures as solid granules rather than viscous liquids. This issue was presciently described by
      • Wilson E.B.
      The structure of protoplasm.
      , who noted the liquid or viscous structures of living starfish oocytes under pressure, positing that (without fixation) intracellular granules and other spherical protoplasmic bodies behaved as a fine emulsion.
      The driving forces behind the assembly and dynamics of membraneless organelles remained largely unexplored for decades. These mechanistic underpinnings came to the forefront in 2009 when the seminal work by
      • Brangwynne C.P.
      • Eckmann C.R.
      • Courson D.S.
      • Rybarska A.
      • Hoege C.
      • Gharakhani J.
      • et al.
      Germline P granules are liquid droplets that localize by controlled dissolution/condensation.
      established that the formation and material properties of P granules were grounded in LLPS dynamics (Figure 1b‒d). They showed that P granules formed in a concentration-dependent manner and exhibited classic liquid-like behaviors of flow under shear stress and rapid fusion into larger droplets (Figure 1c). Building on these observations, they linked P granule assembly to the physics of phase transition polymers, which undergo demixing phase separation to form polymer-rich phases (LLPS condensates) in aqueous solutions. The resulting cross-disciplinary bridge, linking the theoretical framework of LLPS to a broad range of ribonucleoprotein granules, led to a general framework to describe the formation and dynamics (assembly/disassembly, growth, viscosity, buffering capacity) of membraneless organelles (
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ;
      • Jawerth L.
      • Fischer-Friedrich E.
      • Saha S.
      • Wang J.
      • Franzmann T.
      • Zhang X.
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      Protein condensates as aging Maxwell fluids.
      ;
      • Klosin A.
      • Oltsch F.
      • Harmon T.
      • Honigmann A.
      • Jülicher F.
      • Hyman A.A.
      • et al.
      Phase separation provides a mechanism to reduce noise in cells.
      ). Subsequent work with nucleoli (
      • Brangwynne C.P.
      • Mitchison T.J.
      • Hyman A.A.
      Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes.
      ), other ribonucleoprotein assemblies (
      • Garcia-Jove Navarro M.
      • Kashida S.
      • Chouaib R.
      • Souquere S.
      • Pierron G.
      • Weil D.
      • et al.
      RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates.
      ;
      • Sawyer I.A.
      • Sturgill D.M.
      • Dundr M.
      Membraneless nuclear organelles and the search for phases within phases.
      ), and model LLPS condensates (
      • Kaur T.
      • Raju M.
      • Alshareedah I.
      • Davis R.B.
      • Potoyan D.A.
      • Banerjee P.R.
      Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies.
      ) have further delineated this link between LLPS and the liquid-like behaviors of membraneless organelles.
      Over the last decade, a revolution unfolded in pursuit of a biomolecular understanding of LLPS. Going beyond the physicochemistry of synthetic polymers naturally prompted key questions: what biomacromolecules drive LLPS in cells? How is their LLPS controlled at cellular and molecular levels? Initial progress centered on the disordered and aggregation-prone domains of RNA-binding proteins —for their enrichment in prominent ribonucleoprotein granules (
      • Kato M.
      • Han T.W.
      • Xie S.
      • Shi K.
      • Du X.
      • Wu L.C.
      • et al.
      Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
      ). Multidisciplinary efforts eventually converged on IDPs, multivalent protein scaffolds, and RNAs as key players that govern LLPS in the cell (
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ).
      History aside, we turn to key concepts and extant biomolecular LLPS frameworks that illuminate our current understanding of membraneless organelles. First, consider IDPs. Once thought uncommon (
      • Dunker A.K.
      • Lawson J.D.
      • Brown C.J.
      • Williams R.M.
      • Romero P.
      • Oh J.S.
      • et al.
      Intrinsically disordered protein.
      ), IDPs and proteins with IDP domains abound in eukaryotic proteomes (
      • van der Lee R.
      • Buljan M.
      • Lang B.
      • Weatheritt R.J.
      • Daughdrill G.W.
      • Dunker A.K.
      • et al.
      Classification of intrinsically disordered regions and proteins.
      ). IDPs are akin to synthetic polymers, failing to adopt defined three-dimensional (3D) structures to hide away interaction-prone molecular features (Figure 1a) (
      • van der Lee R.
      • Buljan M.
      • Lang B.
      • Weatheritt R.J.
      • Daughdrill G.W.
      • Dunker A.K.
      • et al.
      Classification of intrinsically disordered regions and proteins.
      ). The amino acid composition/sequence of each IDP and the intracellular milieu (solvent) ultimately determine the likelihood, means, and timing of their LLPS behavior. Paralleling observations in synthetic polymers, LLPS leading to condensate formation only occurs when the overall conditions favor intermolecular interactions over IDP-solvent interactions (Figure 1b). Yet, dissecting the intracellular LLPS of IDPs in vivo remains challenging (
      • Alberti S.
      • Gladfelter A.
      • Mittag T.
      Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.
      ). The underlying challenge is two-fold: (i) IDPs span structural features and behaviors intermediate between synthetic polymers and folded proteins and (ii) lack of control/understanding of dynamic cell-specific variables that alter IDPs and their LLPS behavior.
      Borrowing from polymer physics, the LLPS behavior of IDPs may be mapped onto phase diagrams wherein two key variables describe IDP demixing and condensate formation in cells (Figure 1c) (
      • Brangwynne C.P.
      • Tompa P.
      • Pappu R.V.
      Polymer physics of intracellular phase transitions.
      ). The value of this approach relies on the assumption that despite the complexity of the intracellular environment, homotypic interactions dominate the LLPS behavior of a particular IDP. This assumption is reasonable for prototypical LLPS-exhibiting IDPs but may be inadequate for IDPs with LLPS-modifying intracellular binding partners (e.g., RNA for RNA-binding IDPs) and IDPs with low LLPS propensity. The latter may rely on heterotypic cooperation with other IDPs to drive intracellular LLPS (
      • Riback J.A.
      • Zhu L.
      • Ferrolino M.C.
      • Tolbert M.
      • Mitrea D.M.
      • Sanders D.W.
      • et al.
      Composition-dependent thermodynamics of intracellular phase separation.
      ). Phase diagrams for synthetic polymers and IDPs take on different shapes depending on the dominant molecular interactions. The phase diagram depicted in Figure 1c is typical of polymers that only undergo LLPS below an upper critical solution temperature (UCST)—picture the gelation of refrigerated gelatin. Some IDPs exhibit an inverted type of phase diagram defined by a lower critical solution temperature (LCST) (
      • Ambadipudi S.
      • Biernat J.
      • Riedel D.
      • Mandelkow E.
      • Zweckstetter M.
      Liquid –liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau.
      ;
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ;
      • Riback J.A.
      • Katanski C.D.
      • Kear-Scott J.L.
      • Pilipenko E.V.
      • Rojek A.E.
      • Sosnick T.R.
      • et al.
      Stress-triggered phase separation is an adaptive, evolutionarily tuned response.
      ). Characterization of intracellular LLPS has primarily focused on the UCST-type behavior exhibited by oft-studied RNA-binding IDPs (
      • Bracha D.
      • Walls M.T.
      • Wei M.-T.
      • Zhu L.
      • Kurian M.
      • Avalos J.L.
      • et al.
      Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds [published correction appears in Cell 2019;176:407].
      ;
      • Nott T.J.
      • Petsalaki E.
      • Farber P.
      • Jervis D.
      • Fussner E.
      • Plochowietz A.
      • et al.
      Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles.
      ), but IDPs with LCST behavior are well-known from studies of elastin (
      • Brangwynne C.P.
      • Tompa P.
      • Pappu R.V.
      Polymer physics of intracellular phase transitions.
      ;
      • Dzuricky M.
      • Roberts S.
      • Chilkoti A.
      Convergence of artificial protein polymers and intrinsically disordered proteins.
      ;
      • Urry D.W.
      • Long M.M.
      • Cox B.A.
      • Ohnishi T.
      • Mitchell L.W.
      • Jacobs M.
      The synthetic polypentapeptide of elastin coacervates and forms filamentous aggregates.
      ,
      • Urry D.W.
      • Starcher B.
      • Partridge S.M.
      Coacervation of Solubilized elastin effects a notable conformational change.
      ).
      LLPS-exhibiting IDPs are enriched in low complexity and repetitive amino acid motifs that engage in multivalent and weakly adhesive interactions (
      • Alberti S.
      • Gladfelter A.
      • Mittag T.
      Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.
      ;
      • Brangwynne C.P.
      • Tompa P.
      • Pappu R.V.
      Polymer physics of intracellular phase transitions.
      ;
      • Kato M.
      • Han T.W.
      • Xie S.
      • Shi K.
      • Du X.
      • Wu L.C.
      • et al.
      Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
      ). Whether an IDP exhibits LCST or UCST LLPS is specified at the compositional amino acid level, with the molecular features of specific residues influencing the nature of peptide‒peptide interactions (
      • Dignon G.L.
      • Zheng W.
      • Kim Y.C.
      • Mittal J.
      Temperature-controlled liquid–liquid phase separation of disordered proteins.
      ;
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ;
      • Wang J.
      • Choi J.M.
      • Holehouse A.S.
      • Lee H.O.
      • Zhang X.
      • Jahnel M.
      • et al.
      A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins.
      ). These LLPS-specific interactions involve combinations of charge‒charge, cation‒pi, pi‒pi, hydrogen-bonding, and hydrophobic contacts (
      • Das S.
      • Lin Y.H.
      • Vernon R.M.
      • Forman-Kay J.D.
      • Chan H.S.
      Comparative roles of charge, π, and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins.
      ;
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ). Whereas LCST IDPs favor hydrophobic interactions involving aliphatic residues, UCST IDPs often rely on cation‒pi and pi‒pi interactions involving aromatic and arginine residues. Beyond compositional bias, IDPs are sequence-controlled polymers wherein subtle changes at the sequence level potently shift LLPS dynamics (
      • Quiroz F.G.
      • Li N.K.
      • Roberts S.
      • Weber P.
      • Dzuricky M.
      • Weitzhandler I.
      • et al.
      Intrinsically disordered proteins access a range of hysteretic phase separation behaviors.
      ). Although poorly understood, sequence-encoded behaviors set IDPs apart from synthetic polymers. These observations suggest that some membraneless organelles may access unique types of nonequilibrium LLPS dynamics (
      • Jawerth L.
      • Fischer-Friedrich E.
      • Saha S.
      • Wang J.
      • Franzmann T.
      • Zhang X.
      • et al.
      Protein condensates as aging Maxwell fluids.
      ;
      • Quiroz F.G.
      • Li N.K.
      • Roberts S.
      • Weber P.
      • Dzuricky M.
      • Weitzhandler I.
      • et al.
      Intrinsically disordered proteins access a range of hysteretic phase separation behaviors.
      ).
      To intuit the value of temperature-dependent (UCST and LCST) LLPS frameworks in biological systems, consider that temperature is simply a dial of the underlying interaction energies. Changes in pH, ionic strength, and other environmental signals are common dials that either amplify or suppress LLPS, thus shifting the boundaries of the two-phase regime in the relevant phase diagram. Analogously, cells harbor sophisticated molecular dials to tune intermolecular IDP interactions. One major dial involves post-translational modifications (PTMs). Of note, IDPs and disordered protein segments are hotspots for PTMs in eukaryotic cells (
      • Wright P.E.
      • Dyson H.J.
      Intrinsically disordered proteins in cellular signalling and regulation.
      ). As shown in Figure 1e, PTMs such as phosphorylation potently alter the LLPS behavior of IDPs (
      • Owen I.
      • Shewmaker F.
      The role of post-translational modifications in the phase transitions of intrinsically disordered proteins.
      ;
      • Pattanaik A.
      • Gowda D.C.
      • Urry D.W.
      Phosphorylation and dephosphorylation modulation of an inverse temperature transition.
      ). The outcome of these PTMs depends on the molecular features of the PTM and the proteins and the IDPs involved, resulting in either assembly or disassembly of LLPS condensates (
      • Wang J.T.
      • Smith J.
      • Chen B.C.
      • Schmidt H.
      • Rasoloson D.
      • Paix A.
      • et al.
      Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans.
      ). For example, in epidermal differentiation, we suspect that IDP phosphorylation plays a role in KG disassembly. Another dial of intracellular LLPS involves RNAs, which alter LLPS behavior upon interaction with RNA-binding IDPs (
      • Garcia-Jove Navarro M.
      • Kashida S.
      • Chouaib R.
      • Souquere S.
      • Pierron G.
      • Weil D.
      • et al.
      RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates.
      ;
      • Kaur T.
      • Raju M.
      • Alshareedah I.
      • Davis R.B.
      • Potoyan D.A.
      • Banerjee P.R.
      Sequence-encoded and composition-dependent protein-RNA interactions control multiphasic condensate morphologies.
      ;
      • Langdon E.M.
      • Qiu Y.
      • Ghanbari Niaki A.G.
      • McLaughlin G.A.
      • Weidmann C.A.
      • Gerbich T.M.
      • et al.
      mRNA structure determines specificity of a polyQ-driven phase separation.
      ). This is the likely major role for RNAs in tuning intracellular LLPS, although some RNA sequences are themselves prone to LLPS (
      • Jain A.
      • Vale R.D.
      RNA phase transitions in repeat expansion disorders.
      ). Temperature may also directly dial the LLPS behavior of IDPs in cellular contexts subject to environmental extremes. We will discuss this idea for LLPS condensates near the skin surface, but related mechanisms may be at play in other barrier tissues. Temperature fluctuations are also intuitive dials for the LLPS dynamics linked to heat shock responses (
      • Riback J.A.
      • Katanski C.D.
      • Kear-Scott J.L.
      • Pilipenko E.V.
      • Rojek A.E.
      • Sosnick T.R.
      • et al.
      Stress-triggered phase separation is an adaptive, evolutionarily tuned response.
      ) and for biomolecular LLPS in poikilotherms.
      Although the previously introduced biomolecular framework is IDP-centric, valency alone is a major determinant of intracellular LLPS (
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ;
      • Sanders D.W.
      • Kedersha N.
      • Lee D.S.W.
      • Strom A.R.
      • Drake V.
      • Riback J.A.
      • et al.
      Competing protein-RNA interaction networks control multiphase intracellular organization.
      ). In pioneering work by
      • Li P.
      • Banjade S.
      • Cheng H.C.
      • Kim S.
      • Chen B.
      • Guo L.
      • et al.
      Phase transitions in the assembly of multivalent signalling proteins.
      and
      • Banani S.F.
      • Rice A.M.
      • Peeples W.B.
      • Lin Y.
      • Jain S.
      • Parker R.
      • et al.
      Compositional control of phase-separated cellular bodies.
      , Rosen and his team expanded notions of biomolecular LLPS to include networks of interacting multivalent proteins. These studies showed that signaling proteins, which often feature folded repeat domains with small linker IDP segments, engage in heterotypic, multivalent interdomain interactions capable of driving LLPS. Similar to multivalent interactions in IDPs, these domain interactions are weak (low affinity) and are governed by PTMs. However, unlike self-interacting IDPs, PTMs on one multivalent protein alter its binding affinity to domains on other proteins in the network. Because these multivalent repeat proteins often localize to the cell membrane, these studies also uncovered intracellular two-dimensional LLPS condensates—rather than 3D spherical droplets—that stretch abutting the membrane surface (
      • Su X.
      • Ditlev J.A.
      • Hui E.
      • Xing W.
      • Banjade S.
      • Okrut J.
      • et al.
      Phase separation of signaling molecules promotes T cell receptor signal transduction.
      ).
      The expanded biomolecular understanding of LLPS has matured into a useful framework in which membraneless organelles are functionally and compositionally dissected into scaffolds and clients (
      • Banani S.F.
      • Lee H.O.
      • Hyman A.A.
      • Rosen M.K.
      Biomolecular condensates: organizers of cellular biochemistry.
      ). Scaffolds are IDP or multivalent proteins, perhaps even RNAs, which drive intracellular LLPS to form biomolecular condensates. Clients may lack LLPS behavior of their own but accumulate within condensates through client-scaffold interactions. Although these interactions are commonly engineered and conceived as traditional lock-and-key models involving domains in clients and scaffolds (as shown in Figure 1f), the concept extends to ultraweak interactions between IDP scaffolds and IDP clients (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). However, this useful division of labor between clients and scaffolds should not distract from the possibility that their roles intertwine. Across the wide spectrum of potential intracellular clients (
      • van der Lee R.
      • Buljan M.
      • Lang B.
      • Weatheritt R.J.
      • Daughdrill G.W.
      • Dunker A.K.
      • et al.
      Classification of intrinsically disordered regions and proteins.
      ), some client proteins may conditionally exhibit LLPS or modulate the LLPS dynamics of scaffolds and their membraneless organelles (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ;
      • Riback J.A.
      • Zhu L.
      • Ferrolino M.C.
      • Tolbert M.
      • Mitrea D.M.
      • Sanders D.W.
      • et al.
      Composition-dependent thermodynamics of intracellular phase separation.
      ).
      The diversity of mechanisms to encode and control biomolecular LLPS poses a major challenge to predict and study LLPS scaffolds and LLPS clients in the cell. However, this challenge represents an exciting research frontier (
      • Hardenberg M.
      • Horvath A.
      • Ambrus V.
      • Fuxreiter M.
      • Vendruscolo M.
      Widespread occurrence of the droplet state of proteins in the human proteome.
      ). Experimentally, elucidating the evolving material properties and heterogeneous composition of intracellular LLPS condensates remain key areas for progress. Although the integrity of LLPS condensates is typically compromised on tissue and cell lysis, preventing traditional purification, the advent of proximity-dependent proteomics promises to expose the biomolecular composition of membraneless organelles (
      • Bracha D.
      • Walls M.T.
      • Brangwynne C.P.
      Probing and engineering liquid-phase organelles.
      ;
      • Markmiller S.
      • Soltanieh S.
      • Server K.L.
      • Mak R.
      • Jin W.
      • Fang M.Y.
      • et al.
      Context-dependent and disease-specific diversity in protein interactions within stress granules.
      ;
      • Yang P.
      • Mathieu C.
      • Kolaitis R.M.
      • Zhang P.
      • Messing J.
      • Yurtsever U.
      • et al.
      G3BP1 is a tunable switch that triggers phase separation to assemble stress granules.
      ). These approaches, coupled with emergent tools to probe in vivo LLPS without molecular tagging of IDP scaffolds (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ), will fuel progress toward translating the LLPS-imparted dynamics of membraneless organelles into molecular mechanisms in cells and tissues (
      • Dodson A.E.
      • Kennedy S.
      Phase separation in germ cells and development.
      ).

      Membraneless organelles across stratifying epithelia

      Despite the exciting progress in the field of LLPS-driven cellular mechanisms, we still know surprisingly little about the contribution of membraneless organelles and their LLPS dynamics to mammalian and tissue biology. This gap is at full display for abundant membraneless structures in the skin, namely the KGs of the epidermis and the trichohyalin granules (TGs) of hair follicles (Figure 2).
      Figure thumbnail gr2
      Figure 2FLG and paralogs are IDPs that form membraneless organelles across stratifying epithelia. (a) Ultrastructure (TEM) and corresponding schematic of the mouse epidermis. Epidermal progenitor cells reside in the basal layer, attached to a basement membrane (dotted line). Progenitor cells flux upward to the skin surface, gaining KGs (red arrows) in the granular layer, which subsequently disappear as the cells move into the corneum. Adapted from
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      . Bar = 10 μm. (b, c, e, h) Domain architecture of human FLG, RPTN, FLG2, and TCHH and indicated location of disease-linked nonsense mutations (colored lines) as well as recurring nonsense mutations (>10 alleles) that await clinical interrogation (colored triangles) (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alföldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans [published correction appears in Nature 2021;590:E53].
      ). Below the domain architecture, we show the disorder plots that predict the probability of IDP regions (
      • Jones D.T.
      • Cozzetto D.
      DISOPRED3: precise disordered region predictions with annotated protein-binding activity.
      ). A value of 1 indicates disorder. FLG paralogs share a repetitive architecture with N-terminal S100 domains. These S100 domains have conserved hydrophobic pockets but distinct surface chemistries (
      • Hinbest A.J.
      • Kim S.R.
      • Eldirany S.A.
      • Lomakin I.B.
      • Watson J.
      • Ho M.
      • et al.
      Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins.
      ). (b) Mutations in the repeat domain of FLG (labeled as clusters: mut-n0 to mut-n10) generate truncated FLG variants that are strongly associated with atopic dermatitis; adapted from
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      . (d) FLG (pink) and RPTN (green) form distinct immiscible condensates when HaCATs differentiate and stratify at the air‒liquid interface; adapted from
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      . Bar = 5 μm. (e) FLG2 has two domains that exhibit repeat architecture. The A-type repeat domain resembles HRNR repeats, whereas the B-type domain resembles FLG repeats (
      • Wu Z.
      • Hansmann B.
      • Meyer-Hoffert U.
      • Gläser R.
      • Schröder J.M.
      Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.
      ). Truncating mutants of FLG2 are associated with atopic dermatitis in African Americans (
      • Margolis D.J.
      • Gupta J.
      • Apter A.J.
      • Ganguly T.
      • Hoffstad O.
      • Papadopoulos M.
      • et al.
      Filaggrin-2 variation is associated with more persistent atopic dermatitis in African American subjects.
      ) and with Peeling Skin Syndrome (
      • Alfares A.
      • Al-Khenaizan S.
      • Al Mutairi F.
      Peeling skin syndrome associated with novel variant in FLG2 gene.
      ;
      • Mohamad J.
      • Sarig O.
      • Godsel L.M.
      • Peled A.
      • Malchin N.
      • Bochner R.
      • et al.
      Filaggrin 2 deficiency results in abnormal cell-cell adhesion in the cornified cell layers and causes peeling skin syndrome type A.
      ). (f) Analysis of FLG and paralogs in mice, humans, and other mammalian species show that FLG family proteins have conserved sequence features that encode for UCST-type LLPS in IDPs. Adapted from
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ; see the supplementary materials in the study by
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      for a complete list of the studied mammalian species. (g) Ultrastructure (left) and schematic (right) of the organization of a mouse hair follicle. The hair follicle layers are depicted from the deepest to most superficial layers: Me, Co, Ch, Ci, Hu, He, Cp, and ORS. TEM image is courtesy of H. Amalia Pasolli and Elaine Fuchs at Rockefeller University (New York, NY); layer annotations were performed by H. Amalia Pasolli. Schematic of hair follicle layers was adapted from
      • Yang H.
      • Adam R.C.
      • Ge Y.
      • Hua Z.L.
      • Fuchs E.
      Epithelial-mesenchymal micro-niches govern stem cell lineage choices.
      . Bar = 10 μm. (h) TCHH displays a repetitive architecture with five repeat families. This sequence diversity contributes to a ragged disorder profile, in which low-disorder segments are prone to α-helical conformations (
      • Lee S.C.
      • Kim I.G.
      • Marekov L.N.
      • O'keefe E.J.
      • Parry D.A.
      • Steinert P.M.
      The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.
      ). A nonsense mutation at residue 331 is associated with Uncombable Hair Syndrome (
      • Ü Basmanav F.B.
      • Cau L.
      • Tafazzoli A.
      • Méchin M.C.
      • Wolf S.
      • Romano M.T.
      • et al.
      Mutations in three genes encoding proteins involved in hair shaft formation cause uncombable hair syndrome.
      ). AA, amino acid; Arg, arginine; DP, dermal papilla; IDP, intrinsically-disordered protein; KG, keratohyalin granule; LLPS, liquid-liquid phase separation; nu, nucleus; TAC, transit-amplifying cell; TCHH, trichohyalin; TEM, transmission electron microscopy; TG, trichohyalin granule; UCST, upper critical solution temperature; Me, medulla; Co, cortex; Ch, hair shaft cuticle; Ci, inner root sheath cuticle; Hu, Huxley’s layer; He, Henle’s layer; Cp, companion layer; ORS, outer root sheath.
      KGs are the subcellular structures that typify differentiating epidermal cells in the granular layer (
      • Brody I.
      Ultrastructure of the stratum corneum.
      ), disappearing as granular cells flux upward to the stratum corneum (Figure 2a). First visualized by Aufhammer in 1869 and confirmed as a hallmark of epidermal differentiation by Langerhans in 1873 (
      • Matoltsy A.G.
      • Matoltsy M.N.
      The chemical nature of keratohyalin granules of the epidermis.
      ), the enigmatic history of KGs mirrors that of other membraneless organelles (
      • Holbrook K.A.
      Biologic structure and function: perspectives on morphologic approaches to the study of the granular layer keratinocyte.
      ). The term “keratohyalin granules,” coined by Waldeyer in 1882, was inspired by their theorized role as the precursor to keratin—the intracellular substance of corneocytes—and their histological features reminiscent of proteinaceous hyalin (
      • Matoltsy A.G.
      • Matoltsy M.N.
      The chemical nature of keratohyalin granules of the epidermis.
      ). Initially misinterpreted as byproducts of mitochondrial and nuclear degradation or as aggregates of fragmented tonofibrils (an early name for keratin filaments) and even ribosomes, ultrastructural studies with radiolabeled amino acids ultimately established KGs as products of active protein synthesis. Crucially, these studies showed that KGs contained a histidine-rich protein, later identified as FLG (Figure 2b) (
      • Fukuyama K.
      • Nakamura T.
      • Benstein I.A.
      Differentially localized incorporation of amino acids in relation to epidermal keratinization in the newborn rat.
      ), and suggested that their growth involved granule fusion (
      • Brody I.
      The keratinization of epidermal cells of normal guinea pig skin as revealed by electron microscopy.
      ). However, these early efforts often confused FLG-containing KGs with other membraneless structures of the mammalian epidermis, such as the cysteine-rich loricrin granules in rat epidermis (
      • Fukuyama K.
      • Epstein W.L.
      A comparative autoradiographic study of keratogyalin granules containing cystine and histidine.
      ;
      • Holbrook K.A.
      Biologic structure and function: perspectives on morphologic approaches to the study of the granular layer keratinocyte.
      ;
      • Matoltsy A.G.
      • Matoltsy M.N.
      The chemical nature of keratohyalin granules of the epidermis.
      ). While we do not dwell on these pioneering efforts spanning over a century, their careful review (
      • Holbrook K.A.
      Biologic structure and function: perspectives on morphologic approaches to the study of the granular layer keratinocyte.
      ) provides a fascinating window into scientific progress in skin and tissue biology.
      The discovery of FLG as a major constituent of KGs propelled the field of epidermal biology. FLG proteins are large histidine-rich IDPs with a repetitive architecture (Figure 2b), which undergo extensive and species-specific processing throughout epidermal differentiation. We use FLG to refer to the full-length protein encoded by the repetitive gene FLG, irrespective of mammalian species. The goal is to avoid confusion with the historical use of the term filaggrin, which specifically refers to a small basic protein that
      • Dale B.A.
      Purification and characterization of a basic protein from stratum corneum of mammalian epidermis.
      isolated from the corneum of rat epidermis. Later shown to aggregate with purified keratin filaments (
      • Dale B.A.
      • Holbrook K.A.
      • Steinert P.M.
      Assembly of stratum corneum basic protein and keratin filaments in macrofibrils.
      ), the underlying histidine-rich protein was eventually linked to a phosphorylated and oligomerized precursor from rat KGs (
      • Lonsdale-Eccles J.D.
      • Haugen J.A.
      • Dale B.A.
      A phosphorylated keratohyalin-derived precursor of epidermal stratum corneum basic protein.
      ).
      • Steinert P.M.
      • Cantieri J.S.
      • Teller D.C.
      • Lonsdale-Eccles J.D.
      • Dale B.A.
      Characterization of a class of cationic proteins that specifically interact with intermediate filaments.
      subsequently proposed the name filaggrin to describe compositionally conserved small cationic proteins from the corneum of the mammalian epidermis, which they functionally defined by their ability to aggregate purified keratin filaments (
      • Steinert P.M.
      • Cantieri J.S.
      • Teller D.C.
      • Lonsdale-Eccles J.D.
      • Dale B.A.
      Characterization of a class of cationic proteins that specifically interact with intermediate filaments.
      ). When subsequent work established the repetitive rather than oligomerized nature of the unprocessed filaggrin precursor, the term profilaggrin was adopted (
      • Harding C.R.
      • Scott I.R.
      Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.
      ), influencing the pioneering characterization of the human FLG gene (
      • Presland R.B.
      • Haydock P.V.
      • Fleckman P.
      • Nirunsuksiri W.
      • Dale B.A.
      Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.
      ). However, the modern descriptions of this gene (FLG) (
      • Brown S.J.
      • McLean W.H.
      One remarkable molecule: filaggrin.
      ) and protein (FLG) do not attempt to capture how filaggrin led to the discovery of profilaggrin. We adopt and recommend this simplification because the suggested nomenclature adequately shifts focus from specific roles of FLG processing byproducts to the overall functionality of FLG and its KGs.
      The subtle shift from filaggrin to FLG becomes relevant to approach the poorly understood relationships between KG formation/loss and FLG synthesis/processing in skin barrier formation. For example, besides a repeat IDP domain, FLG features an N-terminal calcium-binding and dimerizing S100 domain (
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • Lewis S.P.
      • Lo C.Y.
      • Dale B.A.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ). Long known to be cleaved during epidermal differentiation (
      • Hinbest A.J.
      • Kim S.R.
      • Eldirany S.A.
      • Lomakin I.B.
      • Watson J.
      • Ho M.
      • et al.
      Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins.
      ;
      • Presland R.B.
      • Haydock P.V.
      • Fleckman P.
      • Nirunsuksiri W.
      • Dale B.A.
      Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.
      ), the exact role and fate of this S100 domain remain unclear. Arguably, the only firmly established role of in vivo FLG processing is to provide the prime source of UV-protecting and water-binding free amino acids and amino acid derivatives that keep corneocytes hydrated (
      • Scott I.R.
      • Harding C.R.
      Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.
      ;
      • Scott I.R.
      • Harding C.R.
      • Barrett J.G.
      Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.
      ). Adding to the environmental sensitivity of the skin barrier, this breakdown of FLG fragments in the corneum occurs in a humidity-dependent manner (
      • Scott I.R.
      • Harding C.R.
      Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.
      ). Low humidity promotes the deimination of FLG fragments (
      • Cau L.
      • Pendaries V.
      • Lhuillier E.
      • Thompson P.R.
      • Serre G.
      • Takahara H.
      • et al.
      Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown.
      ), which accelerates their degradation by Caspase-14 (
      • Denecker G.
      • Hoste E.
      • Gilbert B.
      • Hochepied T.
      • Ovaere P.
      • Lippens S.
      • et al.
      Caspase-14 protects against epidermal UVB photodamage and water loss.
      ;
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • Denecker G.
      • Kezic S.
      • Yau N.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ) and bleomycin hydrolase (
      • Kamata Y.
      • Taniguchi A.
      • Yamamoto M.
      • Nomura J.
      • Ishihara K.
      • Takahara H.
      • et al.
      Neutral cysteine protease bleomycin hydrolase is essential for the breakdown of deiminated filaggrin into amino acids.
      ). Disruption of complete FLG processing compromises the hydration and mechanical properties of corneocytes (
      • Thyssen J.P.
      • Jakasa I.
      • Riethmüller C.
      • Schön M.P.
      • Braun A.
      • Haftek M.
      • et al.
      Filaggrin expression and processing deficiencies impair corneocyte surface texture and stiffness in mice.
      ). Several additional proteases contribute to the early stages of FLG processing, including SASPase (ASPRV1) (
      • Matsui T.
      • Miyamoto K.
      • Kubo A.
      • Kawasaki H.
      • Ebihara T.
      • Hata K.
      • et al.
      SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing.
      ), matriptase (ST14) (
      • Alef T.
      • Torres S.
      • Hausser I.
      • Metze D.
      • Türsen U.
      • Lestringant G.G.
      • et al.
      Ichthyosis, follicular atrophoderma, and hypotrichosis caused by mutations in ST14 is associated with impaired profilaggrin processing.
      ;
      • List K.
      • Szabo R.
      • Wertz P.W.
      • Segre J.
      • Haudenschild C.C.
      • Kim S.Y.
      • et al.
      Loss of proteolytically processed filaggrin caused by epidermal deletion of matriptase/MT-SP1.
      ), and prostasin (PRSS8) (
      • Leyvraz C.L.
      • Charles R.P.
      • Rubera I.
      • Guitard M.
      • Rotman S.
      • Breiden B.
      • et al.
      The epidermal barrier function is dependent on the serine protease CAP1/Prss8.
      ;
      • Netzel-Arnett S.
      • Currie B.M.
      • Szabo R.
      • Lin C.Y.
      • Chen L.M.
      • Chai K.X.
      • et al.
      Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation.
      ). However, all the steps before complete FLG degradation, including details of how FLG fragments interact with keratin filaments in the corneum, are incompletely linked to barrier function. For example, disruption of SASPase alters early FLG cleavage products but does not compromise FLG processing to free amino acids, inexplicably reducing corneum hydration and corneocyte stiffness (
      • Matsui T.
      • Miyamoto K.
      • Kubo A.
      • Kawasaki H.
      • Ebihara T.
      • Hata K.
      • et al.
      SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing.
      ;
      • Thyssen J.P.
      • Jakasa I.
      • Riethmüller C.
      • Schön M.P.
      • Braun A.
      • Haftek M.
      • et al.
      Filaggrin expression and processing deficiencies impair corneocyte surface texture and stiffness in mice.
      ). As we will explain, dissecting the overall functionality of FLG and the cellular mechanisms involving its KGs will be key to continued progress in skin barrier research.
      Centuries-old observations linking the absence of KGs to skin pathology (
      • Brody I.
      Ultrastructure of the stratum corneum.
      ;
      • Harding C.R.
      • Scott I.R.
      Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.
      ) continue to fuel fascination with the functional significance of KGs. Although the pioneering work by Unna focused on psoriatic skin (
      • Brody I.
      Ultrastructure of the stratum corneum.
      ), the first molecular links to a defect in FLG synthesis came from families affected by ichthyosis vulgaris (
      • Sybert V.P.
      • Dale B.A.
      • Holbrook K.A.
      Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules.
      ). The latter is the most common inherited disorder of epidermal differentiation, marked by varying degrees of dry and rough skin (
      • Brown S.J.
      • McLean W.H.
      One remarkable molecule: filaggrin.
      ). Yet, the challenge of sequencing the highly repetitive FLG gene long delayed the efforts to uncover the genetic basis of this autosomal semidominant skin disorder (
      • Brown S.J.
      • McLean W.H.
      One remarkable molecule: filaggrin.
      ). Beginning in 2006, refined genotyping strategies firmly established that FLG truncations, namely p.R501X and c.2282del4, cause ichthyosis vulgaris (
      • Smith F.J.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Sandilands A.
      • Campbell L.E.
      • Zhao Y.
      • et al.
      Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.
      ). These two nonsense mutations, which account for >80% of the FLG loss-of-function mutations in Northern Europeans, and others spanning the FLG repeat domain (Figure 2b) are the strongest risk factor for atopic dermatitis (AD) (
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Sandilands A.
      • Terron-Kwiatkowski A.
      • Hull P.R.
      • O'Regan G.M.
      • Clayton T.H.
      • Watson R.M.
      • et al.
      Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.
      ;
      • Wong X.F.C.C.
      • Denil S.L.I.J.
      • Foo J.N.
      • Chen H.
      • Tay A.S.L.
      • Haines R.L.
      • et al.
      Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants.
      ). Highly prevalent among patients with ichthyosis vulgaris and strongly associated with allergies, AD is a common skin inflammatory disorder, a type of eczema in which dry and rough skin may get severely itchy. The FLG mutational landscape varies substantially with ethnicity (
      • Chen H.
      • Ho J.C.
      • Sandilands A.
      • Chan Y.C.
      • Giam Y.C.
      • Evans A.T.
      • et al.
      Unique and recurrent mutations in the filaggrin gene in Singaporean Chinese patients with ichthyosis vulgaris [published correction appears in J Invest Dermatol 2008;128:2545].
      ;
      • Nomura T.
      • Sandilands A.
      • Akiyama M.
      • Liao H.
      • Evans A.T.
      • Sakai K.
      • et al.
      Unique mutations in the filaggrin gene in Japanese patients with ichthyosis vulgaris and atopic dermatitis.
      ;
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ;
      • Zhu Y.
      • Mitra N.
      • Feng Y.
      • Tishkoff S.
      • Hoffstad O.
      • Margolis D.
      FLG variation differs between European Americans and African Americans.
      ). FLG also exhibits intragenic copy (repeat) number variation (between 10 and 12 repeats), and the largest variants are associated with a reduced risk of suffering from skin barrier disorders (
      • Brown S.J.
      • Kroboth K.
      • Sandilands A.
      • Campbell L.E.
      • Pohler E.
      • Kezic S.
      • et al.
      Intragenic copy number variation within filaggrin contributes to the risk of atopic dermatitis with a dose-dependent effect.
      ;
      • Margolis D.J.
      • Mitra N.
      • Berna R.
      • Hoffstad O.
      • Kim B.S.
      • Yan A.
      • et al.
      Associating filaggrin copy number variation and atopic dermatitis in African-Americans: challenges and opportunities.
      ).
      The refined understanding of KGs and FLG was intimately linked to progress in identifying the epidermal differentiation complex (EDC) (
      • Mischke D.
      • Korge B.P.
      • Marenholz I.
      • Volz A.
      • Ziegler A.
      Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex (“epidermal differentiation complex”) on human chromosome 1q21.
      ), a gene cluster in human chromosome 1 that governs terminal differentiation (
      • de Guzman Strong C.
      • Conlan S.
      • Deming C.B.
      • Cheng J.
      • Sears K.E.
      • Segre J.A.
      A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis.
      ;
      • Moreci R.S.
      • Lechler T.
      Epidermal structure and differentiation.
      ). Probing the EDC led to the identification of novel FLG paralogs with an S100-fused type architecture, namely RPTN (
      • Krieg P.
      • Schuppler M.
      • Koesters R.
      • Mincheva A.
      • Lichter P.
      • Marks F.
      Repetin (Rptn), a new member of the “fused gene” subgroup within the S100 gene family encoding a murine epidermal differentiation protein.
      ), HRNR (
      • Makino T.
      • Takaishi M.
      • Morohashi M.
      • Huh N.H.
      Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin.
      ), CRNN, and most recently FLG2 (
      • Wu Z.
      • Hansmann B.
      • Meyer-Hoffert U.
      • Gläser R.
      • Schröder J.M.
      Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.
      ). These FLG paralogs remain understudied without consensus on their tissue specificity, processing, abundance, and functional significance. Except for the unusually small CRNN, their localization is linked to epidermal KGs (
      • Huber M.
      • Siegenthaler G.
      • Mirancea N.
      • Marenholz I.
      • Nizetic D.
      • Breitkreutz D.
      • et al.
      Isolation and characterization of human repetin, a member of the fused gene family of the epidermal differentiation complex.
      ;
      • Wu Z.
      • Hansmann B.
      • Meyer-Hoffert U.
      • Gläser R.
      • Schröder J.M.
      Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.
      ,
      • Wu Z.
      • Meyer-Hoffert U.
      • Reithmayer K.
      • Paus R.
      • Hansmann B.
      • He Y.
      • et al.
      Highly complex peptide aggregates of the S100 fused-type protein hornerin are present in human skin.
      ). These reports hint at the heterogenous composition of KGs, but the evidence remains scarce. RPTN, for example, features a prominent IDP domain (Figure 2c) and can form distinct membraneless granules that interact with FLG-containing KGs (Figure 2d) (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). FLG2 features a long IDP domain with FLG-like repeats (Figure 2e) and is closest to FLG in aspects of in vivo processing and epidermal expression (
      • Hsu C.Y.
      • Henry J.
      • Raymond A.A.
      • Méchin M.C.
      • Pendaries V.
      • Nassar D.
      • et al.
      Deimination of human filaggrin-2 promotes its proteolysis by calpain 1.
      ;
      • Pendaries V.
      • Le Lamer M.
      • Cau L.
      • Hansmann B.
      • Malaisse J.
      • Kezic S.
      • et al.
      In a three-dimensional reconstructed human epidermis filaggrin-2 is essential for proper cornification.
      ). FLG2 is also generally believed to colocalize with FLG in KGs (
      • Hsu C.Y.
      • Henry J.
      • Raymond A.A.
      • Méchin M.C.
      • Pendaries V.
      • Nassar D.
      • et al.
      Deimination of human filaggrin-2 promotes its proteolysis by calpain 1.
      ;
      • Wu Z.
      • Hansmann B.
      • Meyer-Hoffert U.
      • Gläser R.
      • Schröder J.M.
      Molecular identification and expression analysis of filaggrin-2, a member of the S100 fused-type protein family.
      ). Although heterozygous FLG2 truncations are linked to the persistence of AD in African Americans (
      • Margolis D.J.
      • Gupta J.
      • Apter A.J.
      • Ganguly T.
      • Hoffstad O.
      • Papadopoulos M.
      • et al.
      Filaggrin-2 variation is associated with more persistent atopic dermatitis in African American subjects.
      ), FLG and FLG2 may serve nonoverlapping roles in skin barrier formation (
      • Mohamad J.
      • Sarig O.
      • Godsel L.M.
      • Peled A.
      • Malchin N.
      • Bochner R.
      • et al.
      Filaggrin 2 deficiency results in abnormal cell-cell adhesion in the cornified cell layers and causes peeling skin syndrome type A.
      ). For example, homozygous FLG2 truncations are uniquely linked to a skin peeling syndrome (
      • Alfares A.
      • Al-Khenaizan S.
      • Al Mutairi F.
      Peeling skin syndrome associated with novel variant in FLG2 gene.
      ;
      • Mohamad J.
      • Sarig O.
      • Godsel L.M.
      • Peled A.
      • Malchin N.
      • Bochner R.
      • et al.
      Filaggrin 2 deficiency results in abnormal cell-cell adhesion in the cornified cell layers and causes peeling skin syndrome type A.
      ). Unlike FLG, FLG2 shows appreciable localization to the cornified envelope (CE), a hallmark protein-reinforced plasma membrane of corneocytes (
      • Albérola G.
      • Schröder J.M.
      • Froment C.
      • Simon M.
      The amino-terminal part of human FLG2 is a component of cornified envelopes.
      ). Notably, FLG fragments are often cited as contributing to the CE, despite evidence that very small amounts of FLG fragments localize to the CE (
      • Manabe M.
      • Sanchez M.
      • Sun T.T.
      • Dale B.A.
      Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris.
      ;
      • Simon M.
      • Haftek M.
      • Sebbag M.
      • Montézin M.
      • Girbal-Neuhauser E.
      • Schmitt D.
      • et al.
      Evidence that filaggrin is a component of cornified cell envelopes in human plantar epidermis.
      ;
      • Yoneda K.
      • Hohl D.
      • McBride O.W.
      • Wang M.
      • Cehrs K.U.
      • Idler W.W.
      • et al.
      The human loricrin gene.
      ). The significant variance in sequence and architecture across FLG and FLG paralogs suggest specialized roles in epidermal biology, but their association with KGs or distinct membraneless condensates likely influences their functionality. One striking observation that insinuates functional LLPS capabilities is that FLG and its paralogs all share key compositional determinants of UCST-type behavior (Figure 2f) (
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ;
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ).
      Soon after the early studies of KGs in the epidermis, the hyalin of the hair drew intense interest. In 1903, Voerner first reported TGs in cells of the inner root sheath and medulla layers of hair (Figure 2g) (
      • Hamilton E.H.
      • Payne Jr., R.E.
      • O'keefe E.J.
      Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis.
      ). Reminiscent of KG dynamics, the formation of subcellular TGs hallmarks distinct stages of hair follicle differentiation, disappearing abruptly as cells undergo terminal differentiation (
      • Birbeck M.
      • Mercer E.H.
      The electron microscopy of the human hair follicle, III. The inner root sheath and trichohyaline.
      ;
      • O'Keefe E.J.
      • Hamilton E.H.
      • Lee S.C.
      • Steinert P.
      Trichohyalin: a structural protein of hair, tongue, nail, and epidermis.
      ). Similar to KGs, TGs exist in close association with keratin filaments, and their formation and processing are convincingly yet incompletely linked to the high-order structuring of the keratin network (
      • O'Guin W.M.
      • Sun T.T.
      • Manabe M.
      Interaction of trichohyalin with intermediate filaments: three immunologically defined stages of trichohyalin maturation.
      ). Some early descriptions of TG dynamics suggested that they behaved as viscous insoluble liquids, coalescing to form larger droplets before their sudden disappearance (
      • Birbeck M.
      • Mercer E.H.
      The electron microscopy of the human hair follicle, III. The inner root sheath and trichohyaline.
      ). The major known protein component of TGs is TCHH, a large IDP-like protein (Figure 2h) first isolated from wool follicles (
      • Rothnagel J.A.
      • Rogers G.E.
      Trichohyalin, an intermediate filament-associated protein of the hair follicle.
      ). Similar to FLG, the TCHH gene was a founding member of the EDC complex (
      • Mischke D.
      • Korge B.P.
      • Marenholz I.
      • Volz A.
      • Ziegler A.
      Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex (“epidermal differentiation complex”) on human chromosome 1q21.
      ) and features an FLG-like repetitive architecture (
      • Lee S.C.
      • Kim I.G.
      • Marekov L.N.
      • O'keefe E.J.
      • Parry D.A.
      • Steinert P.M.
      The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.
      ). Apart from having an S100 domain fused with a long repetitive segment, the amino acid sequence and composition of TCHH are strikingly different from those of FLG (
      • Lee S.C.
      • Kim I.G.
      • Marekov L.N.
      • O'keefe E.J.
      • Parry D.A.
      • Steinert P.M.
      The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.
      ;
      • Rothnagel J.A.
      • Rogers G.E.
      Trichohyalin, an intermediate filament-associated protein of the hair follicle.
      ). Figure 2h shows the ragged disorder profile of TCHH, with some repeat segments having a high alpha-helical propensity (
      • Lee S.C.
      • Kim I.G.
      • Marekov L.N.
      • O'keefe E.J.
      • Parry D.A.
      • Steinert P.M.
      The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.
      ). TCHH is one of the most enriched in charged residues across the human proteome (
      • Lee S.C.
      • Kim I.G.
      • Marekov L.N.
      • O'keefe E.J.
      • Parry D.A.
      • Steinert P.M.
      The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.
      ), with arginine and glutamine accounting for nearly 50% of its composition. TCHH has long been shown to undergo extensive citrullination (
      • Rogers G.E.
      Isolation and properties of inner sheath cells of hair follicles.
      ;
      • Rothnagel J.A.
      • Rogers G.E.
      Trichohyalin, an intermediate filament-associated protein of the hair follicle.
      ), which has been implicated in TG dissolution and transglutaminase-mediated crosslinking of TCHH with itself and with keratins (
      • Tarcsa E.
      • Marekov L.N.
      • Andreoli J.
      • Idler W.W.
      • Candi E.
      • Chung S.I.
      • et al.
      The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases.
      ). These crosslinking events are considered the primary mechanism by which TCHH mechanically strengthens terminally differentiated inner root sheath cells (
      • Steinert P.M.
      • Parry D.A.
      • Marekov L.N.
      Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth.
      ).
      Beyond epidermal and hair differentiation, KGs and TGs recur in terminal differentiation programs across stratifying epithelia. TGs feature prominently in the filiform papillae of the tongue (
      • O'Keefe E.J.
      • Hamilton E.H.
      • Lee S.C.
      • Steinert P.
      Trichohyalin: a structural protein of hair, tongue, nail, and epidermis.
      ), whereas KGs are abundant in the epithelium of the hard palate (
      • De Benedetto A.
      • Qualia C.M.
      • Baroody F.M.
      • Beck L.A.
      Filaggrin expression in oral, nasal, and esophageal mucosa.
      ;
      • Smith S.A.
      • Dale B.A.
      Immunologic localization of filaggrin in human oral epithelia and correlation with keratinization.
      ). Similar to our observation of interacting KGs and RPTN granules (Figure 2d), immunogold labeling of tongue epithelium has revealed intriguing interactions between KGs and TGs (
      • Manabe M.
      • O'Guin W.M.
      Existence of trichohyalin-keratohyalin hybrid granules: co-localization of two major intermediate filament-associated proteins in non-follicular epithelia.
      ). A subset of cells in the granular layer of the human epidermis contain TGs (
      • Hamilton E.H.
      • Payne Jr., R.E.
      • O'keefe E.J.
      Trichohyalin: presence in the granular layer and stratum corneum of normal human epidermis.
      ), but their frequency and significance remain unexplored. However, as we explain next, how KGs and TGs contribute to terminal differentiation begs intense exploration.

      Liquid-liquid phase separation dynamics through skin barrier formation

      Understanding FLG proteins as prototypical LLPS-exhibiting IDP scaffolds redefined the formation and disappearance of KGs as barrier-defining LLPS dynamics (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Using live imaging and genetically engineered mice with epidermal expression of phase-separation sensors,
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      uncovered the FLG-dependent LLPS-driven assembly, maturation, and disassembly of KGs. Figure 3a summarizes our current understanding of in vivo LLPS dynamics through epidermal stratification, as epidermal cells flux into the granular layer and through the granule-to-corneum transition.
      Figure thumbnail gr3
      Figure 3LLPS dynamics through epidermal stratification. (a) Shifting LLPS dynamics of KGs play a prominent role throughout epidermal stratification. On detachment from the BM, keratinocytes acquire keratin filaments formed by keratin-1/ keratin-10 (K1/K10) heterodimers. Moving upward, keratinocytes upregulate FLG, triggering the LLPS-driven assembly of submicron KGs (stage 1), which later become prominently visible (≥1 μm in diameter) in the granular layer where FLG levels are highest. As cells move through the granular layer (stages 2 and 3), KGs grow in number and volume and become closely associated with keratin bundles as they crowd the cytoplasm. At the granular‒corneum interface, one granular cell (top right) undergoes pH-triggered KG disassembly and nuclear compaction as it moves upward to the corneum. At the corneum layer, free of KGs, nuclei, and other organelles, corneocytes are filled with a highly ordered network of keratin filaments (
      • Iwai I.
      • Han H.
      • den Hollander L.
      • Svensson S.
      • Ofverstedt L.G.
      • Anwar J.
      • et al.
      The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.
      ). Under direct environmental stress, corneocytes at the skin surface eventually slough off, either cooperating with or responding to the underlying epidermal dynamics. The vertical gradient scales highlight the progressive crowding of the cytoplasm by KGs (cyan) and keratin bundles (gray). (b‒d) Inspired by live imaging of KG-phase dynamics in mouse skin (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ), these panels depict KG-phase dynamics inside a single keratinocyte as it fluxes through the granular layer (stages 1‒3, time points t1 through t6). (b) In the early stages of KG assembly, KGs are not caged by keratin bundles (t1), move freely, and exhibit liquid-like fusion events (t2‒t3) that fuel their early growth. (c) As FLG levels rise and KGs become prominent (t4), they are well-distributed in the cytoplasm, and keratin cages prevent their liquid-like fusion. With sustained FLG synthesis over the span of 1‒2 days (t4‒t5), KGs grow substantially in volume as FLG molecules flow from the dilute phase (cytoplasm) to the KG condensates. This KG growth is accompanied by an increase in their viscosity. As viscous KGs grow, keratin bundles and other organelles are compacted in the increasingly crowded cytoplasm. Suddenly (t6), KGs begin to disassemble and release their contents to drive corneocyte formation. (d) KG-phase dynamics are pH sensitive. Immediately before cornification, the cell experiences an abrupt intracellular acidification that initiates KG disassembly. (e) In some mature granular layer cells (stage 3), viscous KGs prominently deform nuclei (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). When FLG truncation mutants do not abolish LLPS (e.g., mut-n8 and tail-mut in b), these FLG mutants form less viscous KGs that wet the nuclear surface (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). (f) pH-triggered KG dissolution releases yet unknown KG components that actuate rapid enucleation, beginning with prominent chromatin compaction and quickly leading to organelle loss and squame features. BM, basement membrane; KG, keratohyalin granule; LLPS, liquid-liquid phase separation.
      KGs in early granular-layer cells occasionally grow through liquid-like fusion events (Figure 3b). However, as KCs move upward toward the skin surface, they become crowded with increasingly viscous KGs that grow primarily without fusion (Figure 3c). Departing from the extensive liquid-like fusion of de novo assembled KGs in cultured KCs, abundant in vivo keratin-1/keratin-10 (K1/K10) fibers interact with FLG on the surface of native KGs to cage them, preventing KG fusion (Figure 3c) (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Through electron microscopy, these keratin-caged KGs appear prominently on horizontal, en face sections of mouse epidermis (
      • Usui K.
      • Kadono N.
      • Furuichi Y.
      • Shiraga K.
      • Saitou T.
      • Kawasaki H.
      • et al.
      3D in vivo imaging of the keratin filament network in the mouse stratum granulosum reveals profilaggrin-dependent regulation of keratin bundling.
      ). Furthermore, this restriction of KG fusion by K1/K10 fibers likely controls KG size, as suggested by enlarged KGs on genetic ablation of K10 in mice (
      • Kumar V.
      • Bouameur J.E.
      • Bär J.
      • Rice R.H.
      • Hornig-Do H.T.
      • Roop D.R.
      • et al.
      A keratin scaffold regulates epidermal barrier formation, mitochondrial lipid composition, and activity [published correction appears in J Cell Biol 2016;212:877].
      ). In humans with epidermolytic hyperkeratosis characterized by KRT1 and KRT10 mutations, K1/K10 aggregates severely disrupt the overall cytoplasmic organization and result in unusually large KGs (
      • Ishida-Yamamoto A.
      • Eady R.A.
      • Underwood R.A.
      • Dale B.A.
      • Holbrook K.A.
      Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis).
      ). The emergent picture suggests that a tug-of-war between KGs and keratin fibers structures the cytoplasm en route to cornification.
      Coinciding with the granular-to-corneum transition (the top-right granular cell in Figure 3a), KGs begin to disassemble (Figure 3c). This sudden shift in KG-phase dynamics is triggered by an abrupt drop in intracellular pH (Figure 3d). The pH responsiveness of KGs is rooted in the pH-sensitive LLPS behavior of FLG, which becomes protonated as the intracellular pH approaches the pka (~pH 6.1) of its abundant histidine residues (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Although the skin surface has been long known to be acidic, suggesting an extracellular pH gradient across the corneum layer, live imaging of KG-residing phase-separation sensors and intracellular pH reporters uncovered a novel intracellular pH shift as an upstream regulator of the granular-to-corneum transition (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). In complementary intravital imaging,
      • Matsui T.
      • Kadono-Maekubo N.
      • Suzuki Y.
      • Furuichi Y.
      • Shiraga K.
      • Sasaki H.
      • et al.
      A unique mode of keratinocyte death requires intracellular acidification.
      showed that this critical and rapid intracellular acidification occurs downstream of a tightly regulated and sustained increase in intracellular calcium levels. Before KG disassembly, viscous KGs distort nuclei (Figure 3e) and possibly other membrane-bound organelles. These KG-induced nuclear deformations contrast with the wetting of nuclear surfaces by P granules (
      • Wei M.T.
      • Elbaum-Garfinkle S.
      • Holehouse A.S.
      • Chen C.C.
      • Feric M.
      • Arnold C.B.
      • et al.
      Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.
      ), suggesting that KGs are biomolecular condensates with distinct mechanical/viscous properties.
      Crucial to skin barrier formation, pH-triggered KG disassembly initiates enucleation (Figure 3f). Combined live imaging of phase-separation sensors, chromatin, and intracellular pH reporters in mouse skin showed that upon the abrupt intracellular acidification, the release of KG-residing client proteins precedes chromatin compaction, leading to enucleation and squame-like features (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). In the early stages of enucleation, KG disassembly and intracellular acidification may facilitate nuclear entry and activation of DNase1L2 (
      • Fischer H.
      • Buchberger M.
      • Napirei M.
      • Tschachler E.
      • Eckhart L.
      Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis.
      ,
      • Fischer H.
      • Eckhart L.
      • Mildner M.
      • Jaeger K.
      • Buchberger M.
      • Ghannadan M.
      • et al.
      DNase1L2 degrades nuclear DNA during corneocyte formation.
      ;
      • Matsui T.
      • Kadono-Maekubo N.
      • Suzuki Y.
      • Furuichi Y.
      • Shiraga K.
      • Sasaki H.
      • et al.
      A unique mode of keratinocyte death requires intracellular acidification.
      ), which resides in the endoplasmic reticulum (ER) (
      • Fischer H.
      • Szabo S.
      • Scherz J.
      • Jaeger K.
      • Rossiter H.
      • Buchberger M.
      • et al.
      Essential role of the keratinocyte-specific endonuclease DNase1L2 in the removal of nuclear DNA from hair and nails.
      ) and is unique among DNase I family members in its dual dependence on calcium and acidic pH (active at pH ≤6.4) (
      • Shiokawa D.
      • Tanuma S.
      Characterization of human DNase I family endonucleases and activation of DNase gamma during apoptosis.
      ). Importantly, when manipulating intracellular acidification of the granular layer, pH-triggered chromatin compaction occurred in a KG-dependent manner (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). In granular layer cells genetically depleted of KGs, the process of enucleation lost its characteristic rapid progression, and intracellular acidification failed to trigger chromatin compaction (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Adding to these findings in mice,
      • Ipponjima S.
      • Umino Y.
      • Nagayama M.
      • Denda M.
      Live imaging of alterations in cellular morphology and organelles during cornification using an epidermal equivalent model.
      , working with human skin equivalents, showed that loss of FLG and KGs drastically lengthened the rapid morphological transformations underlying the granular-to-corneum transition.
      That the enigmatic granular-to-corneum transition entails a pH-triggered shift in epidermal LLPS dynamics provides new biomolecular insights to probe the long-known association between absence/atypical KGs and cornification defects in the human epidermis (
      • Brody I.
      Ultrastructure of the stratum corneum.
      ;
      • Manabe M.
      • Sanchez M.
      • Sun T.T.
      • Dale B.A.
      Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris.
      ). To guide progress, initial efforts should map the differentiation and pH-dependent molecular composition of KGs, identifying KG clients that mediate enucleation/cornification and the enzymes involved in controlling KG disassembly. The latter is intriguing because given the high pH buffering capacity of large histidine-rich KGs, intracellular pH shifts are insufficient to complete KG disassembly. Nonetheless, intracellular acidification alters KG-phase dynamics, resulting in the partial release of FLG scaffold and prominent release of KG clients into the cytoplasm, while allowing entry of proteins previously excluded from KGs (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). To offer an example, in genetically engineered mice with epidermal expression of SEpHLuorin (a genetically encoded pH reporter), KGs exclude the pH reporter and only become enriched in SEpHLuorin upon the pH-triggered onset of KG disassembly—also revealing pH buffering inside of KGs (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). The underlying pH-triggered change in KG permeability likely exposes FLG scaffolds to PTMs, which further alter its LLPS behavior to favor KG disassembly.
      Regarding PTMs of FLG, the prevailing notion is that FLG is rapidly phosphorylated on synthesis, before KG formation, and that FLG dephosphorylation occurs upstream of KG dissolution and FLG processing at the granular-to-corneum transition (
      • Brown S.J.
      • McLean W.H.
      One remarkable molecule: filaggrin.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ). This widely held view is at odds with the discovery that LLPS-specific homotypic FLG‒FLG interactions drive KG assembly. Considering epidermal LLPS dynamics, we suspect that the phosphorylation of FLG contributes to KG disassembly rather than to assembly. In line with this LLPS-inspired model, early attempts to transiently express FLG fragments in cultured cells showed that intracellular formation of KG-like structures does not involve FLG phosphorylation (
      • Dale B.A.
      • Presland R.B.
      • Lewis S.P.
      • Underwood R.A.
      • Fleckman P.
      Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.
      ). Arguably, the current understanding of FLG as heavily phosphorylated has roots in the language barrier separating FLG and filaggrin. Whereas this purified FLG fragment appeared unphosphorylated in corneum extracts, larger FLG fragments from the granular layer appeared phosphorylated (
      • Lonsdale-Eccles J.D.
      • Teller D.C.
      • Dale B.A.
      Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.
      ,
      • Lonsdale-Eccles J.D.
      • Haugen J.A.
      • Dale B.A.
      A phosphorylated keratohyalin-derived precursor of epidermal stratum corneum basic protein.
      ). When purified filaggrin was eventually linked to profilaggrin in KGs, phosphorylation was assigned to the earliest occurrence of detectable FLG before KG assembly (
      • Lonsdale-Eccles J.D.
      • Teller D.C.
      • Dale B.A.
      Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.
      ,
      • Lonsdale-Eccles J.D.
      • Haugen J.A.
      • Dale B.A.
      A phosphorylated keratohyalin-derived precursor of epidermal stratum corneum basic protein.
      ). Complicating this context, subsequent characterization of FLG and FLG fragments relied on their pelleting from tissue lysates through dilution in ice-cold water—equivalent to IDP purification through thermally triggered LLPS (
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ). Given the UCST-type LLPS behavior of FLG, this simple purification strategy likely skewed against phosphorylated FLG fragments (
      • Resing K.A.
      • Walsh K.A.
      • Dale B.A.
      Identification of two intermediates during processing of profilaggrin to filaggrin in neonatal mouse epidermis.
      ), cementing the belief that phosphorylation is somehow specific to newly synthesized FLG. However, to our knowledge, we lack convincing evidence to link this phosphorylated precursor of filaggrin to KG-residing FLG in early granular-layer cells. Instead, we submit that the phosphorylated precursor derives from cells at the granular-to-corneum interface, where FLG phosphorylation may be at play in KG disassembly.
      This LLPS-inspired model for phosphorylation-dependent KG disassembly has important implications. First, the model is consistent with observations that phosphorylation of FLG fragments prevents premature FLG-mediated keratin aggregation (
      • Harding C.R.
      • Scott I.R.
      Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.
      ;
      • Lonsdale-Eccles J.D.
      • Teller D.C.
      • Dale B.A.
      Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.
      ). Before KG disassembly, condensate-restricted FLG‒keratin interactions occur and are beneficial to structuring the cytoplasm (Figure 3c). Second, the model is in line with recent phosphoproteomic data on UV-irradiated human skin equivalents (
      • Yang F.
      • Waters K.M.
      • Webb-Robertson B.J.
      • Sowa M.B.
      • von Neubeck C.
      • Aldrich J.T.
      • et al.
      Quantitative phosphoproteomics identifies filaggrin and other targets of ionizing radiation in a human skin model.
      ). Third, the model invites the search for kinases capable of mediating FLG phosphorylation and KG disassembly at the granular-to-corneum transition. Finally, as originally suspected for rat FLG, subsequent proteolytic processing of FLG may occur in a phosphorylation-dependent manner (
      • Resing K.A.
      • Johnson R.S.
      • Walsh K.A.
      Characterization of protease processing sites during conversion of rat profilaggrin to filaggrin.
      ). KG disassembly and FLG processing may also be modulated by intracellular acidification at the granule-to-corneum transition by SASPase (
      • Matsui T.
      • Kadono-Maekubo N.
      • Suzuki Y.
      • Furuichi Y.
      • Shiraga K.
      • Sasaki H.
      • et al.
      A unique mode of keratinocyte death requires intracellular acidification.
      ), which becomes active at pH ≤ 6.5 (
      • Matsui T.
      • Kinoshita-Ida Y.
      • Hayashi-Kisumi F.
      • Hata M.
      • Matsubara K.
      • Chiba M.
      • et al.
      Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation.
      ).
      Although several proteases contribute to processing FLG fragments to free amino acids, the early stages of FLG processing that are most relevant to shifting KG-phase dynamics remain poorly understood (
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • Denecker G.
      • Kezic S.
      • Yau N.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ;
      • List K.
      • Szabo R.
      • Wertz P.W.
      • Segre J.
      • Haudenschild C.C.
      • Kim S.Y.
      • et al.
      Loss of proteolytically processed filaggrin caused by epidermal deletion of matriptase/MT-SP1.
      ;
      • Matsui T.
      • Miyamoto K.
      • Kubo A.
      • Kawasaki H.
      • Ebihara T.
      • Hata K.
      • et al.
      SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ). Even the timing and role of the well-known cleavage of the N-terminal S100 domain of FLG remain unclear. However, this event has been typically assumed to occur at the granular-to-corneum transition, after KG dissolution (
      • Cabanillas B.
      • Novak N.
      Atopic dermatitis and filaggrin.
      ;
      • Eckhart L.
      • Lippens S.
      • Tschachler E.
      • Declercq W.
      Cell death by cornification.
      ;
      • Hoste E.
      • Kemperman P.
      • Devos M.
      • Denecker G.
      • Kezic S.
      • Yau N.
      • et al.
      Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.
      Filaggrin in the frontline: role in skin barrier function and disease.
      ). We recently learned that the S100 domain of FLG, which undergoes calcium-independent dimerization (
      • Bunick C.G.
      • Presland R.B.
      • Lawrence O.T.
      • Pearton D.J.
      • Milstone L.M.
      • Steitz T.A.
      Crystal structure of human profilaggrin S100 domain and identification of target proteins annexin II, stratifin, and HSP27.
      ), strongly promotes KG formation by reducing the critical concentration for intracellular LLPS of FLG (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      )—equivalent to CDilute in Figure 1d. In engineered FLG proteins lacking S100 processing, the resulting intracellular KGs showed unusual stiffening, suggesting that early removal of the S100 domain may be required to sustain the liquid-like dynamics of KGs. Closing this knowledge gap, the composition of KGs in genetically engineered mice with epidermal expression of GFP fused to the S100 domain of mouse FLG (mS100-GFP) agrees with this early timing for S100 removal. In cultured KCs, the KGs assembled from unprocessed FLG prominently accumulate mS100-GFP, likely through S100-mediated dimerization. In contrast, native KGs in cells of the middle and late granular layers of mouse epidermis prominently excluded mS100-GFP (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). These in vivo data point to the removal of the S100 domain soon after KG assembly, with the S100 domain primarily facilitating KG formation. This model is consistent with reports showing that HRNR and extracted full-length FLG react poorly with antibodies directed to the S100 domain (
      • Presland R.B.
      • Kimball J.R.
      • Kautsky M.B.
      • Lewis S.P.
      • Lo C.Y.
      • Dale B.A.
      Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.
      ;
      • Wu Z.
      • Meyer-Hoffert U.
      • Reithmayer K.
      • Paus R.
      • Hansmann B.
      • He Y.
      • et al.
      Highly complex peptide aggregates of the S100 fused-type protein hornerin are present in human skin.
      ).
      Under the lens of LLPS, establishing KG-phase dynamics is a primary role for FLG. The axiom follows that FLG nonsense mutations must interfere with KG assembly and KG maturation. In line with this view, quantitative live-cell imaging of fluorescently tagged FLG mutants showed that truncation of the FLG repeat domain potently alters the critical concentration for intracellular LLPS. Full-length FLG (with 12 FLG repeats) readily undergoes LLPS at low intracellular levels (~2 μM). In contrast, common FLG truncation mutants (with ≤4 FLG repeats) essentially lose the ability to undergo intracellular LLPS, exhibiting exceedingly high critical concentrations for LLPS (~130 to >1,500 μM) (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Before the onset of skin inflammation, FLG nonsense mutations do not severely alter FLG mRNA transcripts (
      • Nirunsuksiri W.
      • Presland R.B.
      • Brumbaugh S.G.
      • Dale B.A.
      • Fleckman P.
      Decreased profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.
      ) or prevent their translation (
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      ;
      • Manabe M.
      • Sanchez M.
      • Sun T.T.
      • Dale B.A.
      Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris.
      ;
      • Sandilands A.
      • Terron-Kwiatkowski A.
      • Hull P.R.
      • O'Regan G.M.
      • Clayton T.H.
      • Watson R.M.
      • et al.
      Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.
      ). Yet, KG assembly is consistently compromised, emphasizing the repeat-encoded LLPS of FLG. Losing their ability to undergo LLPS at low intracellular levels, FLG truncated mutants likely follow the fate of many soluble IDPs, becoming targets for degradation (
      • van der Lee R.
      • Buljan M.
      • Lang B.
      • Weatheritt R.J.
      • Daughdrill G.W.
      • Dunker A.K.
      • et al.
      Classification of intrinsically disordered regions and proteins.
      ). Intriguingly, common nonsense FLG mutations that largely spare the repeat domain are also associated with AD (Figure 2b). Although FLG variants with >8 tandem FLG repeats drive KG assembly at low critical concentrations, removal of the tiny C-terminal tail domain (drawn to scale in Figure 2b)—a puzzling 26-residue sequence conserved across mammals (
      • Presland R.B.
      • Haydock P.V.
      • Fleckman P.
      • Nirunsuksiri W.
      • Dale B.A.
      Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.
      ;
      • Sandilands A.
      • Sutherland C.
      • Irvine A.D.
      • McLean W.H.
      Filaggrin in the frontline: role in skin barrier function and disease.
      )— decreases KG viscosity, switching KGs from nuclei deforming to nuclei wetting (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ).
      These LLPS-inspired observations suggest that the fine tuning of KG viscosity contributes to skin barrier formation. Notably, despite the lack of sequence conservation between mouse and human FLG, they share LLPS-specific compositional biases (Figure 2f) and assemble KGs of similar viscosity (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Contrary to expectation, de novo assembled KGs in cultured, poorly differentiated KCs are less viscous than native KGs in stratifying epidermis, suggesting that differentiation-associated proteins modulate in vivo KG-phase dynamics (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Revisiting FLG nonsense mutations, mutants with increasingly larger truncations of the IDP domain, if permissive for KG assembly (e.g., downstream of mut-n4 in Figure 2b), assemble KG-like structures with progressive downward shifts in viscosity (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). A prediction follows that progressively N-terminal FLG mutations may lead to more severe skin barrier phenotypes. However, current genetic studies largely oversimplify FLG truncation as loss of function (
      • Margolis D.J.
      • Mitra N.
      • Wubbenhorst B.
      • D’Andrea K.
      • Kraya A.A.
      • Hoffstad O.
      • et al.
      Association of filaggrin loss-of-function variants with race in children with atopic dermatitis.
      ).
      This novel focus on KG-phase dynamics informs a new perspective to understand the role of FLG in skin barrier formation and its mutational landscape in skin barrier disorders. Arguably, the analysis of FLG truncations remains heavily influenced by the original focus on filaggrin. Mutations are interpreted and quantified as loss of processed FLG repeats in the corneum. A priori, capturing the disease severity of FLG genotypes requires the lens of epidermal LLPS dynamics. This view is relevant to interpreting skin phenotypes in humans with compound heterozygous FLG mutations, combining alleles with N-terminal and C-terminal FLG truncations (
      • Sandilands A.
      • Terron-Kwiatkowski A.
      • Hull P.R.
      • O'Regan G.M.
      • Clayton T.H.
      • Watson R.M.
      • et al.
      Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.
      ). Similarly, although the protective effect of long FLG variants has been typically analyzed as a total sum of available FLG repeats across the two alleles, the lowest risk is already conferred by having one 12-repeat FLG allele (
      • Margolis D.J.
      • Mitra N.
      • Berna R.
      • Hoffstad O.
      • Kim B.S.
      • Yan A.
      • et al.
      Associating filaggrin copy number variation and atopic dermatitis in African-Americans: challenges and opportunities.
      ). We propose that these complex genotype‒phenotype relationships are rooted in the strong repeat-encoded LLPS behaviors of FLG proteins, which dictate KG-phase dynamics. As FLG genotyping continues to improve and expands beyond nonsense mutations (
      • Margolis D.J.
      • Mitra N.
      • Wubbenhorst B.
      • Nathanson K.L.
      Filaggrin sequencing and bioinformatics tools.
      ,
      • Margolis D.J.
      • Mitra N.
      • Wubbenhorst B.
      • D’Andrea K.
      • Kraya A.A.
      • Hoffstad O.
      • et al.
      Association of filaggrin loss-of-function variants with race in children with atopic dermatitis.
      ;
      • Zhu Y.
      • Mitra N.
      • Feng Y.
      • Tishkoff S.
      • Hoffstad O.
      • Margolis D.
      FLG variation differs between European Americans and African Americans.
      ), we suggest refining the definition of FLG loss-of-function mutations to account for their impact on epidermal LLPS dynamics.

      Outlook

      The central role of KG-phase dynamics in skin barrier formation was presciently described by
      • Dale B.A.
      • Resing K.A.
      • Presland R.B.
      Keratohyalin granule proteins.
      when they wrote that “packing and unpacking of keratohyalin granules, and the biochemical changes in the constituent proteins, will be revealed to be exquisitely adapted to this controlled cellular reorganization.” Realizing this vision, through the lens of epidermal LLPS dynamics, KGs emerge as liquid-like membraneless organelles whose assembly and disassembly fuel skin barrier formation (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ).
      Building on this conceptual and experimental progress, our knowledge of KG-phase dynamics suggests new questions and approaches to crack the skin barrier at cellular and molecular levels. Traditionally studied as a cell death mechanism, the granular-to-corneum transition does not accomplish cell death per se, but the formation of the all-important functional skin barrier (
      • Eckhart L.
      • Lippens S.
      • Tschachler E.
      • Declercq W.
      Cell death by cornification.
      ;
      • Koenig U.
      • Robenek H.
      • Barresi C.
      • Brandstetter M.
      • Resch G.P.
      • Gröger M.
      • et al.
      Cell death induced autophagy contributes to terminal differentiation of skin and skin appendages.
      ). This mechanistic and functional distinction strongly motivates the adoption of corneoptosis (
      • Matsui T.
      • Kadono-Maekubo N.
      • Suzuki Y.
      • Furuichi Y.
      • Shiraga K.
      • Sasaki H.
      • et al.
      A unique mode of keratinocyte death requires intracellular acidification.
      ) as the preferred term to describe the early stages of corneocyte formation. Beyond a renewed focus on FLG mutations and profiling of KG clients through proximity-dependent proteomics, we propose four major directions to link LLPS dynamics with concurrent cellular processes at play in corneoptosis.
      First, because environmental extremes uniquely act on the skin surface, we envision that future work will uncover the links between epidermal LLPS dynamics and the environmental resilience of the skin barrier. Because this resilience is lacking in humans with KG defects, these efforts may illuminate therapeutic avenues to address skin barrier disorders. As an example, cold temperatures and low humidity are linked to exacerbation of skin phenotypes in humans with AD (
      • Engebretsen K.A.
      • Johansen J.D.
      • Kezic S.
      • Linneberg A.
      • Thyssen J.P.
      The effect of environmental humidity and temperature on skin barrier function and dermatitis.
      ). Temperature fluctuations are relevant because the LLPS behavior of FLG-like IDPs is highly temperature sensitive (
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ), and cells in the granular layer may experience skin temperature gradients. We suspect that temperature-dependent KG-phase dynamics contribute to the environmental responsiveness of the skin barrier.
      Second, understanding the role of intracellular crowding will be critical to capturing the remarkable structural reorganizations associated with KG assembly, maturation, and disassembly. Macromolecular crowding of the intracellular space is emerging as a biophysical means of tuning cellular mechanisms (
      • Delarue M.
      • Brittingham G.P.
      • Pfeffer S.
      • Surovtsev I.V.
      • Pinglay S.
      • Kennedy K.J.
      • et al.
      mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding.
      ;
      • Mourão M.A.
      • Hakim J.B.
      • Schnell S.
      Connecting the dots: the effects of macromolecular crowding on cell physiology.
      ). In a crowded cellular environment, the interaction between membraneless organelles and membrane-bound organelles merits attention. For example, in osteosarcoma cells in culture, ER exit sites closely interact with membraneless P-bodies to control their fusion and fission (
      • Lee J.E.
      • Cathey P.I.
      • Wu H.
      • Parker R.
      • Voeltz G.K.
      Endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles.
      ). In the skin, these potential organelle interactions may contribute to the overall cytoplasmic organization imposed by maturing KGs (Figure 3c) and its subsequent remodeling during KG dissolution. Abundant KGs may influence the morphology and function of the ER and other organelles as epidermal cells approach the granular-to-corneum transition. For example, potential KG‒ER interactions could link the pH-triggered and KG-dependent dynamics of enucleation to the release of DNase1L2 from the ER. KG-induced crowding may serve as one of the missing stratification-specific regulators of the selective autophagic degradation and eventual loss of mitochondria (
      • Simpson C.L.
      • Tokito M.K.
      • Uppala R.
      • Sarkar M.K.
      • Gudjonsson J.E.
      • Holzbaur E.L.F.
      NIX initiates mitochondrial fragmentation via DRP1 to drive epidermal differentiation.
      ). Similarly, crowding with organized arrays of KGs may contribute to the scaffolding of the tubulo-reticular trans-Golgi network that mediates the barrier-defining secretion of membrane-bound lamellar bodies (
      • Elias P.M.
      • Cullander C.
      • Mauro T.
      • Rassner U.
      • Kömüves L.
      • Brown B.E.
      • et al.
      The secretory granular cell: the outermost granular cell as a specialized secretory cell.
      ;
      • Yamanishi H.
      • Soma T.
      • Kishimoto J.
      • Hibino T.
      • Ishida-Yamamoto A.
      Marked changes in lamellar granule and trans-Golgi network structure occur during epidermal keratinocyte differentiation.
      ). In addition to organelle interactions, KG crowding appears to influence the higher-order bundling of keratins (Figure 3c). Although the puzzling association between KGs and keratin fibers has long been documented through electron microscopy (
      • Brody I.
      An ultrastructural study on the role of the keratohyalin granules in the keratinization process.
      ), new experimental insights showed that IDP domains in KRT1 and KRT10 bind to FLG in KGs (
      • Quiroz F.G.
      • Fiore V.F.
      • Levorse J.M.
      • Polak L.
      • Wong E.
      • Pasolli H.A.
      • et al.
      Liquid-liquid phase separation drives skin barrier formation.
      ). Unable to enter FLG-rich KGs, K1/K10 fibers organize around KGs. We surmise that KG-interacting keratin fibers further pack and bundle as growing arrays of KGs exclude a larger volume of the cytoplasm. In line with this view, keratin network defects are already evident in the granular layer of Flg-null mice (
      • Kawasaki H.
      • Nagao K.
      • Kubo A.
      • Hata T.
      • Shimizu A.
      • Mizuno H.
      • et al.
      Altered stratum corneum barrier and enhanced percutaneous immune responses in filaggrin-null mice.
      ;
      • Usui K.
      • Kadono N.
      • Furuichi Y.
      • Shiraga K.
      • Saitou T.
      • Kawasaki H.
      • et al.
      3D in vivo imaging of the keratin filament network in the mouse stratum granulosum reveals profilaggrin-dependent regulation of keratin bundling.
      ). The collective evidence points to a role for growing KGs in orchestrating progressive keratin assembly before FLG processing and corneoptosis. Future work should re-examine how KG disassembly, involving FLG processing and KG clients, impacts keratin bundling and reorganization of keratin fibers in the early corneocyte matrix (
      • Iwai I.
      • Han H.
      • den Hollander L.
      • Svensson S.
      • Ofverstedt L.G.
      • Anwar J.
      • et al.
      The human skin barrier is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety.
      ).
      Third, uncovering the key molecular modulators of KG maturation and KG disassembly may offer new nodes to understand skin barrier formation and targets to address barrier disorders. The role of PTMs in governing KG disassembly is an obvious target for future research. The role of specific kinases in KG-phase dynamics deserves particular attention, but FLG proteolysis and arginine deimination through PADI3 are also relevant (
      • Méchin M.C.
      • Enji M.
      • Nachat R.
      • Chavanas S.
      • Charveron M.
      • Ishida-Yamamoto A.
      • et al.
      The peptidylarginine deiminases expressed in human epidermis differ in their substrate specificities and subcellular locations.
      ;
      • Nachat R.
      • Méchin M.C.
      • Takahara H.
      • Chavanas S.
      • Charveron M.
      • Serre G.
      • et al.
      Peptidylarginine deiminase isoforms 1–3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin.
      ). The latter may also be at play in tuning the material properties of TGs (
      • Ü Basmanav F.B.
      • Cau L.
      • Tafazzoli A.
      • Méchin M.C.
      • Wolf S.
      • Romano M.T.
      • et al.
      Mutations in three genes encoding proteins involved in hair shaft formation cause uncombable hair syndrome.
      ;
      • Tarcsa E.
      • Marekov L.N.
      • Andreoli J.
      • Idler W.W.
      • Candi E.
      • Chung S.I.
      • et al.
      The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases.
      ). Regarding KG maturation, the potential role of FLG paralogs as drivers or modulators of epidermal LLPS behavior represents an exciting avenue for exploration, especially within the context of skin barrier disorders (
      • Rahrig S.
      • Dettmann J.M.
      • Brauns B.
      • Lorenz V.N.
      • Buhl T.
      • Kezic S.
      • et al.
      Transient epidermal barrier deficiency and lowered allergic threshold in filaggrin-hornerin (FlgHrnr−/−) double-deficient mice.
      ). Additional IDPs in the skin may also exhibit LLPS behavior. To offer a notable example, loricrin is a prototypical glycine-rich IDP (
      • Candi E.
      • Schmidt R.
      • Melino G.
      The cornified envelope: a model of cell death in the skin.
      ) akin to LCST-exhibiting IDPs (
      • Quiroz F.G.
      • Chilkoti A.
      Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers.
      ). Loricrin changes substantially in length across species, concurrently shifting in subcellular localization. Mouse loricrin (486 residues) forms distinct membraneless granules in the granular layer but not in the human epidermis where loricrin (312 residues) resides in FLG-rich KGs (
      • Yoneda K.
      • Hohl D.
      • McBride O.W.
      • Wang M.
      • Cehrs K.U.
      • Idler W.W.
      • et al.
      The human loricrin gene.
      ). Given the prominent role of loricrin in epidermal differentiation (
      • Candi E.
      • Schmidt R.
      • Melino G.
      The cornified envelope: a model of cell death in the skin.
      ), future work should examine how loricrin influences KG-phase dynamics and the reverse (how KGs impact loricrin function) in the human epidermis. Moreover, current efforts to explore FLG upregulation (
      • Otsuka A.
      • Doi H.
      • Egawa G.
      • Maekawa A.
      • Fujita T.
      • Nakamizo S.
      • et al.
      Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression.
      ) and FLG fragments as therapeutic strategies (
      • Cabanillas B.
      • Novak N.
      Atopic dermatitis and filaggrin.
      ;
      • Stout T.E.
      • McFarland T.
      • Mitchell J.C.
      • Appukuttan B.
      • Timothy Stout J.
      Recombinant filaggrin is internalized and processed to correct filaggrin deficiency.
      ) may benefit from considering and targeting KG-phase dynamics.
      Fourth, moving beyond KG-phase dynamics, epidermal biology may uniquely exploit intracellular LLPS dynamics at cell‒cell junctions. We are intrigued by the recent discovery that tight junction proteins assemble through LLPS (
      • Beutel O.
      • Maraspini R.
      • Pombo-García K.
      • Martin-Lemaitre C.
      • Honigmann A.
      Phase separation of zonula occludens proteins drives formation of tight junctions.
      ). Although tight junction assembly plays a critical role in the skin barrier, the details of how epidermal cells restrict its assembly to the uppermost granular layer remain elusive. We also lack an understanding of how these junctions respond dynamically to environmental pressures on the skin barrier (
      • Rübsam M.
      • Mertz A.F.
      • Kubo A.
      • Marg S.
      • Jüngst C.
      • Goranci-Buzhala G.
      • et al.
      E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning.
      ). Another example involves membraneless desmoplakin-containing particles in the assembly of epidermal desmosomes, which consist of dense protein clusters that anchor keratins to the plasma membranes at intercellular junctions (
      • Godsel L.M.
      • Hsieh S.N.
      • Amargo E.V.
      • Bass A.E.
      • Pascoe-McGillicuddy L.T.
      • Huen A.C.
      • et al.
      Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.
      ). The study of such biomolecular condensates at cell‒cell junctions (
      • Belardi B.
      • Son S.
      • Felce J.H.
      • Dustin M.L.
      • Fletcher D.A.
      Cell–cell interfaces as specialized compartments directing cell function.
      ) is an exciting direction for future research at the interface of LLPS and skin biology.
      Finally, we foresee broad exploration of LLPS dynamics across epithelial terminal differentiation programs. Dissection of TG-phase dynamics represents an important direction for unraveling the remarkably complex process of terminal differentiation in hair formation. Given the prominent role of TGs in this process and the emergent genetic links between TCHH mutations and hair disorders (
      • Ü Basmanav F.B.
      • Cau L.
      • Tafazzoli A.
      • Méchin M.C.
      • Wolf S.
      • Romano M.T.
      • et al.
      Mutations in three genes encoding proteins involved in hair shaft formation cause uncombable hair syndrome.