Filaggrin and paralogs enable resilient intracellular liquid-liquid phase separation dynamics in the skin

Summary: Abstract Body: Intracellular liquid-liquid phase separation (LLPS) and related biomolecular condensates govern wide-ranging cellular mechanisms. We recently uncovered that the keratohyalin granules (KGs) of the epidermis are liquid-like condensates whose Filaggrin (FLG)-driven assembly and pH-triggered disassembly propel terminal differentiation during skin barrier formation. Disease-linked FLG truncation potently abrogates intracellular LLPS, providing new insights into the longstanding link between FLG mutations and skin barrier disorders. FLG and its paralogs are gigantic intrinsically-disordered repeat proteins that have long eluded study through conventional genetic and biophysical approaches. Their repeat domains differ at the sequence level but share related LLPS-promoting compositional features. While FLG2 and RPTN are close in composition to FLG, highly-divergent TCHH defies extant LLPS heuristics. Here, applying cutting-edge biomolecular engineering and live-cell imaging approaches to the entire FLG paralog family, we probed the intracellular LLPS of full-length paralogs and intriguing FLG2 variants. Excitingly, we succeeded in cloning full-length TCHH and RPTN and discovered that both readily undergo intracellular LLPS in human keratinocytes. Like KGs, TCHH condensates fused extensively, suggesting that the trichohyalin granules of hair follicles are liquid-like condensates. Both TCHH and RPTN condensates recruited FLG-like proteins, reminiscent of hybrid epidermal granules. FLG2-like proteins also formed liquid-like condensates. Surprisingly, unlike FLG truncation, frequent FLG2 nonsense mutations favored condensate assembly. Tracing this phenotype to the unique two-domain architecture of FLG2, A-type (HRNR-like) repeats potently drove LLPS while B-type (FLG-like) repeats dampened LLPS. Our findings expose resilient mechanisms to impart functional LLPS dynamics across FLG and paralogs, cementing the skin as a prime tissue system to uncover condensate biology. Alexa Regina C. Avecilla¹, Mariell Joy Pascual¹, Felipe G. Quiroz¹ 1. Biomedical Engineering, Wallace H Coulter Department of Biomedical Engineering, Atlanta, GA, United States. Epidermal Structure and Barrier Function