Orchestrated control of filaggrin–actin scaffolds underpins cornification

Abstract

Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier.

Addressing differentiation in live human keratinocytes by assessment of membrane packing order

“…Membrane order assessment in live human epidermal keratinocytes at single-cell level. Previously, we extensively characterised the coordinated role of actin scaffolds and filaggrin granule formation during differentiation of normal human epidermal keratinocytes (NHEKs) (9). Specifically, we monitored filaggrin and actin at different calcium concentrations and described and quantified the distribution of these supramolecular assemblies in cornification.…”

“…Ratiometrically measuring the emission intensities at 440 and 490nm renders the Laurdan "general polarization index" (GP), which allows relative quantification of the membrane order or degree of lipid lateral packing (30,31,35). This method has been used in visualizing native membrane microdomains in planar supported bilayers, giant unilamellar vesicles (36,37) and in living cells (9,(38)(39)(40)(41). Even though the membrane order can only be measured indirectly, and being aware that the stiffness of a membrane influences can be in principle better characterised by fluorescence polarisation or fluorescence anisotropy, in this paper we introduced membrane lateral packing or order as a readout for keratinocyte differentiation advancement.…”

“…related to the cell shape and adherence) and distinct organellar transformations. For instance, the appearance and maturation of keratohyalin granules (KHGs) (9), nuclear condensation and extrusion (10,11), cytoskeleton collapse (9), and alterations in mitochondria (12); in some of those events, autophagy has been shown to be involved (13). At the molecular level, shift in cytokeratin expression has been documented (keratin 5, keratin 14 are progressively replaced with keratin 1 and keratin 10) while additional proteins are upregulated, e.g.…”

Live imaging of alterations in cellular morphology and organelles during cornification using an epidermal equivalent model

“…Actin filaments are also known to be responsible for distributing organelles and mitochondrial depolarization [41][42][43] . Moreover, Gutowska-Owsiak et al showed that actin formed structures associated with filaggrin-containing keratohyalin granules and suggested that actin disruption accelerates keratinocyte differentiation programmes by filaggrin release from keratohyalin granules 44 . Therefore, the series of morphological changes we observed may result from www.nature.com/scientificreports www.nature.com/scientificreports/ actin cytoskeleton collapse.…”

The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains

“…pEGFP-C1-MKLP1 (Addgene plasmid 70145) (Douglas, et al, 2010), which was a kind Gräfelfing, Germany) were stained with 40 µM Laurdan for 30 min at 37°C or 5 µM di-4-ANEPPDHQ just before imaging. After excitation at λ405 nm (Gutowska-Owsiak, et al, 2018;Sezgin, et al, 2015), Laurdan intensity images were simultaneously recorded with emission in the ranges of λ420-460 nm and λ470-510 nm, which were referred to as λ440 and λ490 nm, respectively. For experiments with di-4-ANEPPDHQ, cells were excited at λ488 nm and the emission in the ranges of λ500-580 nm and λ620-750 nm was collected.…”