Science - USA (2020-03-13)

4. M. Fericet al., Coexisting liquid phases underlie nucleolar
   subcompartments.Cell 165 , 1686–1697 (2016).
   doi:10.1016/j.cell.2016.04.047;pmid:27212236


5. X. Suet al., Phase separation of signaling molecules
   promotes T cell receptor signal transduction.Science 352 ,
   595 – 599 (2016). doi:10.1126/science.aad9964;
   pmid: 27056844


6. J. T. Wanget al., Regulation of RNA granule dynamics by
   phosphorylation of serine-rich, intrinsically disordered proteins
   in C. elegans.eLife 3 , e04591 (2014). doi:10.7554/eLife.04591;
   pmid: 25535836


7. A. Molliexet al., Phase separation by low complexity domains
   promotes stress granule assembly and drives pathological
   fibrillization.Cell 163 , 123–133 (2015). doi:10.1016/
   j.cell.2015.09.015; pmid: 26406374


8. B. R. Sabariet al., Coactivator condensation at super-
   enhancers links phase separation and gene control.
   Science 361 , eaar3958 (2018). doi:10.1126/science.aar3958;
   pmid: 29930091


9. W.-K. Choet al., Mediator and RNA polymerase II clusters
   associate in transcription-dependent condensates.
   Science 361 , 412–415 (2018). doi:10.1126/science.aar4199;
   pmid: 29930094


10. B. A. Gibsonet al., Organization of chromatin by intrinsic and
    regulated phase separation.Cell 179 , 470–484.e21 (2019).
    doi:10.1016/j.cell.2019.08.037; pmid: 31543265


11. G. Wanet al., Spatiotemporal regulation of liquid-like
    condensates in epigenetic inheritance.Nature 557 , 679– 683
    (2018). doi:10.1038/s41586-018-0132-0; pmid: 29769721


12. Y. Lin, D. S. Protter, M. K. Rosen, R. Parker, Formation and
    maturation ofphase-separatedliquid droplets by RNA-binding
    proteins.Mol. Cell 60 , 208–219 (2015). doi:10.1016/
    j.molcel.2015.08.018; pmid: 26412307


13. H. Jianget al., Phase transition of spindle-associated protein
    regulate spindle apparatus assembly.Cell 163 , 108– 122
    (2015). doi:10.1016/j.cell.2015.08.010; pmid: 26388440


14. A. K. Rai, J.-X. Chen, M. Selbach, L. Pelkmans, Kinase-
    controlled phase transition of membraneless organelles in
    mitosis.Nature 559 , 211–216 (2018). doi:10.1038/s41586-018-
    0279-8; pmid: 29973724


15. O. Beutel, R. Maraspini, K. Pombo-García, C. Martin-Lemaitre,
    A. Honigmann, Phase Separation of Zonula Occludens Proteins
    Drives Formation of Tight Junctions.Cell 179 , 923–936.e11
    (2019). doi:10.1016/j.cell.2019.10.011; pmid: 31675499


16. E. S. Freeman Rosenzweiget al., The eukaryotic CO2-
    concentrating organelle is liquid-like and exhibits dynamic
    reorganization.Cell 171 ,148–162.e19 (2017). doi:10.1016/
    j.cell.2017.08.008; pmid: 28938114


17. S. Alberti, A. Gladfelter, T. Mittag, Considerations and
    Challenges in Studying Liquid-Liquid Phase Separation and
    Biomolecular Condensates.Cell 176 , 419–434 (2019).
    doi:10.1016/j.cell.2018.12.035; pmid: 30682370


18. H. B. Schmidt, A. Barreau, R. Rohatgi, Phase separation-
    deficient TDP43 remains functional in splicing.Nat. Commun.
    10 , 4890 (2019). doi:10.1038/s41467-019-12740-2;
    pmid: 31653829


19. D. Brachaet al., Mapping Local and Global Liquid Phase
    Behavior in Living Cells Using Photo-Oligomerizable Seeds.Cell
    175 , 1467–1480.e13 (2018). doi:10.1016/j.cell.2018.10.048;
    pmid: 30500534


20. J. A. Segre, Epidermal barrier formation and recovery in skin
    disorders.J. Clin. Invest. 116 , 1150–1158 (2006). doi:10.1172/
    JCI28521;pmid:^16670755
    21.B. A. Dale, K. A. Resing, R. B. Presland, inThe Keratinocyte
    Handbook, I. Leigh, E. B. Lane, F. M. Watt, Eds. (Cambridge
    Univ. Press, 1994), chap. 17, pp. 323–350.


21. F. G. Quiroz, A. Chilkoti, Sequence heuristics to encode phase
    behaviour in intrinsically disordered protein polymers.
    Nat. Mater. 14 , 1164–1171 (2015). doi:10.1038/nmat4418;
    pmid: 26390327


22. C. N. Palmeret al., Common loss-of-function variants of the
    epidermal barrier protein filaggrin are a major predisposing
    factor for atopic dermatitis.Nat. Genet. 38 , 441–446 (2006).
    doi:10.1038/ng1767; pmid: 16550169


23. S. J. Brown, W. H. McLean, One remarkable molecule: Filaggrin.
    J. Invest. Dermatol. 132 , 751–762 (2012). doi:10.1038/
    jid.2011.393; pmid: 22158554


24. D. J. Margoliset al., Filaggrin-2 variation is associated with
    more persistent atopic dermatitis in African American subjects.
    J. Allergy Clin. Immunol. 133 , 784–789 (2014). doi:10.1016/
    j.jaci.2013.09.015; pmid: 24184149


25. S. Rahriget al., Transient epidermal barrier deficiency and
    lowered allergic threshold in filaggrin-hornerin (FlgHrnr-/-)

double-deficient mice.Allergy 74 , 1327–1339 (2019).

doi:10.1111/all.13756; pmid: 30828807

27. X. F. C. C. Wonget al., Array-based sequencing of filaggrin
    gene for comprehensive detection of disease-associated
    variants.J. Allergy Clin. Immunol. 141 ,814–816 (2018).
    doi:10.1016/j.jaci.2017.10.001; pmid: 29056476


28. C.-A. Loet al., Quantification of protein levels in single living
    cells.Cell Rep. 13 , 2634–2644 (2015). doi:10.1016/
    j.celrep.2015.11.048; pmid: 26686644
    29.C.G.Bunicket al., Crystal structure of human
    profilaggrin S100 domain and identification of target
    proteins annexin II, stratifin, and HSP27.J. Invest. Dermatol.
    135 ,1801–1809 (2015). doi:10.1038/jid.2015.102;
    pmid: 25760235


29. S. F. Bananiet al., Compositional control of phase-separated
    cellular bodies.Cell 166 , 651–663 (2016). doi:10.1016/
    j.cell.2016.06.010; pmid: 27374333


30. B. S. Schusteret al., Controllable protein phase separation and
    modular recruitment to form responsive membraneless
    organelles.Nat. Commun. 9 , 2985 (2018). doi:10.1038/
    s41467-018-05403-1; pmid: 30061688


31. T. Christensen, W. Hassouneh, K. Trabbic-Carlson, A. Chilkoti,
    Predicting transition temperatures of elastin-like polypeptide
    fusion proteins.Biomacromolecules 14 , 1514–1519 (2013).
    doi:10.1021/bm400167h; pmid: 23565607


32. B. R. McNaughton, J. J. Cronican, D. B. Thompson, D. R. Liu,
    Mammalian cell penetration, siRNA transfection, and DNA
    transfection by supercharged proteins.Proc. Natl. Acad.
    Sci. U.S.A. 106 , 6111–6116 (2009). doi:10.1073/
    pnas.0807883106; pmid: 19307578


33. J. Jaubert, S. Patel, J. Cheng, J. A. Segre, Tetracycline-
    regulated transactivatorsdrivenby the involucrin promoter to
    achieve epidermal conditional gene expression.J. Invest.
    Dermatol. 123 , 313–318 (2004). doi:10.1111/j.0022-
    202X.2004.23203.x; pmid: 15245431


34. C. Bonnartet al., Elastase 2 is expressed in human and mouse
    epidermis and impairs skin barrier function in Netherton
    syndrome through filaggrin and lipid misprocessing.
    J. Clin. Invest. 120 , 871–882 (2010). doi:10.1172/JCI41440;
    pmid: 20179351


35. P. Rompolaset al., Spatiotemporal coordination of stem cell
    commitment during epidermal homeostasis.Science 352 ,
    1471 – 1474 (2016). doi:10.1126/science.aaf7012;
    pmid: 27229141


36. T.Kartasova,D.R.Roop,K.A.Holbrook,S.H.Yuspa,Mouse
    differentiation-specific keratins 1 and 10 require a
    preexisting keratin scaffold to form a filament network.
    J. Cell Biol. 120 , 1251–1261 (1993). doi:10.1083/
    jcb.120.5.1251;pmid:7679677


37. V. Kumaret al., A keratin scaffold regulates epidermal barrier
    formation, mitochondrial lipid composition, and activity.J. Cell
    Biol. 211 , 1057–1075 (2015). doi:10.1083/jcb.201404147;
    pmid: 26644517


38. C.-H. Lee, M.-S. Kim, B. M. Chung, D. J. Leahy,
    P. A. Coulombe, Structural basis for heteromeric assembly
    and perinuclear organization of keratin filaments.Nat.
    Struct.Mol.Biol. 19 ,707–715 (2012). doi:10.1038/
    nsmb.2330;pmid:22705788


39. M. P. Hugheset al., Atomic structures of low-complexity
    protein segments reveal kinkedbsheets that assemble
    networks.Science 359 , 698–701 (2018). doi:10.1126/science.
    aan6398; pmid: 29439243


40. S. Nodaet al., The Asian atopic dermatitis phenotype
    combines features of atopic dermatitis and psoriasis with
    increased TH17 polarization.J. Allergy Clin. Immunol. 136 ,
    1254 – 1264 (2015).doi: 10 .1016/j.jaci.2015.08.015;
    pmid: 26428954


41. T. J. Nottet al., Phase transition of a disordered nuage protein
    generates environmentally responsive membraneless
    organelles.Mol. Cell 57 , 936–947 (2015). doi:10.1016/
    j.molcel.2015.01.013; pmid: 25747659


42. M. Dzuricky, S. Roberts, A. Chilkoti, Convergence of artificial
    protein polymers and intrinsically disordered proteins.
    Biochemistry 57 , 2405–2414 (2018). doi:10.1021/acs.
    biochem.8b00056; pmid: 29683665


43. R. Niesneret al., 3D-resolved investigation of the pH gradient
    in artificial skin constructs by means of fluorescence lifetime
    imaging.Pharm. Res. 22 , 1079–1087 (2005). doi:10.1007/
    s11095-005-5304-6; pmid: 16028008


44. J. A. Mackay, D. J. Callahan, K. N. Fitzgerald, A. Chilkoti,
    Quantitative model of the phase behavior of recombinant
    pH-responsive elastin-like polypeptides.Biomacromolecules 11 ,
    2873 – 2879 (2010). doi:10.1021/bm100571j; pmid: 20925333
    46. S. Alberti, Guilty by association: Mapping out the
    molecular sociology of droplet compartments.Mol. Cell 69 ,
    349 – 351 (2018). doi:10.1016/j.molcel.2018.01.020;
    pmid: 29395058
    47. S. Markmilleret al., Context-dependent and disease-specific
    diversity in protein interactions within stress granules.
    Cell 172 , 590–604.e13 (2018). doi:10.1016/j.cell.2017.12.032;
    pmid: 29373831
    48. I. Brody, An ultrastructural study on the role of the
    keratohyalin granules in the keratinization process.
    J. Ultrastruct. Res. 3 ,84–104 (1959). doi:10.1016/S0022-5320
    (59)80018-6; pmid: 13804667
    49. T. Makino, M. Takaishi, M. Morohashi, N. H. Huh, Hornerin, a
    novel profilaggrin-like protein and differentiation-specific
    marker isolated from mouse skin.J. Biol. Chem. 276 ,
    47445 – 47452 (2001).doi: 10 .1074/jbc.M107512200;
    pmid: 11572870
    50. P. M. Steinert, D. A. Parry, L. N. Marekov, Trichohyalin
    mechanically strengthens the hair follicle: Multiple cross-
    bridging roles in the inner root shealth.J. Biol. Chem. 278 ,
    41409 – 41419 (2003). doi:10.1074/jbc.M302037200;
    pmid: 12853460
    51. B. Mészároset al., PhaSePro: The database of proteins
    driving liquid–liquid phase separation.Nucleic Acids Res.
    48 ,D360–D367 (2020). doi:10.1093/nar/gkz848;
    pmid: 31612960
    52. J. Kyte, R. F. Doolittle, A simple method for displaying the
    hydropathic character of a protein.J. Mol. Biol. 157 ,105– 132
    (1982). doi:10.1016/0022-2836(82)90515-0;
    pmid: 7108955
    53. J. R. McDaniel, J. A. Mackay, F. G. Quiroz, A. Chilkoti,
    Recursive directional ligation by plasmid reconstruction
    allows rapid and seamless cloning of oligomeric genes.
    Biomacromolecules 11 , 944–952 (2010). doi:10.1021/
    bm901387t;pmid: 20184309
    54. A. C. Woerneret al., Cytoplasmic protein aggregates interfere
    with nucleocytoplasmic transport of protein and RNA.
    Science 351 , 173–176 (2016). doi:10.1126/science.aad2033;
    pmid: 26634439
    55. S. P. Boudkoet al., Crystal structure of human collagen XVIII
    trimerization domain: A novel collagen trimerization Fold.
    J. Mol. Biol. 392 , 787–802 (2009). doi:10.1016/
    j.jmb.2009.07.057; pmid: 19631658
    56. A. Ghoorchian, N. B. Holland, Molecular architecture influences
    the thermally induced aggregation behavior of elastin-like
    polypeptides.Biomacromolecules 12 , 4022–4029 (2011).
    doi:10.1021/bm201031m; pmid: 21972921
    57. N. Maas-Szabowski, A. Stärker, N. E. Fusenig, Epidermal tissue
    regeneration and stromal interaction in HaCaT cells is initiated
    by TGF-a.J. CellSci. 116 , 2937 – 2948 (2003). doi:10.1242/
    jcs.00474; pmid: 12771184
    58. J. A. Nowak, E. Fuchs, inStem Cells in Regenerative Medicine.
    (Springer, 2009), pp. 215-232.
    59. S. Beronja, G. Livshits, S. Williams, E. Fuchs, Rapid functional
    dissection of genetic networks via tissue-specific transduction
    and RNAi in mouse embryos.Nat. Med. 16 , 821–827 (2010).
    doi:10.1038/nm.2167; pmid: 20526348
    60. S. Sankaranarayanan, D. De Angelis, J. E. Rothman, T. A. Ryan,
    The use of pHluorins for optical measurements of presynaptic
    activity.Biophys. J. 79 , 2199–2208 (2000). doi:10.1016/
    S0006-3495(00)76468-X; pmid: 11023924
    61. D. E. Johnsonet al., Red fluorescent protein pH biosensor to
    detect concentrative nucleoside transport.J. Biol. Chem. 284 ,
    20499 – 20511 (2009). doi:10.1074/jbc.M109.019042;
    pmid: 19494110

ACKNOWLEDGMENTS

We thank I. Matos for assistance with the live-imaging

setup; L. Hidalgo and M. Sribour for animal assistance; and

S. Ellis and I. Matos for discussion and comments on the

manuscript. We thank the Molecular Cytology Core Facility at

MSKCC and Rockefeller University (RU) Bio-Imaging

Resource Center for use of microscopes, and RU Comparative

Bioscience Center (AAALAC-accredited) for care of mice

in accordance with National Institutes of Health (NIH)

guidelines.Funding:This work was funded by NIH (R01-

AR27883 to E.F.) and partly supported by the Robertson

Therapeutic Development Fund and by a Tri-Institutional Starr

Stem Cell Scholars Fellowship at The Rockefeller University.

E.F. is an HHMI investigator. F.G.Q. holds a Career Award

at the Scientific Interface from Burroughs Wellcome Fund.

Author contributions:F.G.Q. and E.F. conceived the project,

designed the experiments, and interpreted the data. F.G.Q. and

Quirozet al.,Science 367 , eaax9554 (2020) 13 March 2020 11 of 12

RESEARCH | RESEARCH ARTICLE