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Abstract
Differentiation of keratinocytes is critical for epidermal stratification and formation of a protective stratum corneum. It involves a series of complex processes leading through gradual changes in characteristics and functions of keratinocytes up to their programmed cell death via cornification. The stratum corneum is a relatively impermeable barrier, comprised of dead cell remnants (corneocytes) embedded in lipid matrix. Corneocyte membranes are comprised of specialized lipids linked to late differentiation proteins, contributing to the formation of a stiff and mechanically strengthened layer. To date, the assessment of the progression of keratinocyte differentiation is only possible through determination of specific differentiation markers, e.g., by using proteomics-based approaches. Unfortunately, this requires fixation or cell lysis, and currently there is no robust methodology available to study keratinocyte differentiation in living cells in real-time. Here, we explore new live-cell based approaches for screening differentiation advancement in keratinocytes, in a “calcium switch” model. We employ a polarity-sensitive dye, Laurdan, and Laurdan general polarization function (GP) as a reporter of the degree of membrane lateral packing order or condensation, as an adequate marker of differentiation. We show that the assay is straightforward and can be conducted either on a single cell level using confocal spectral imaging or on the ensemble level using a fluorescence plate reader. Such systematic quantification may become useful for understanding mechanisms of keratinocyte differentiation, such as the role of membrane in homogeneities in stiffness, and for future therapeutic development.
Highlights
The epidermis, in the form of a stratified and desquamating tissue is a fascinating feature of many organisms; it retains very distinct characteristics which enable it to exert function, shielding us from external threats and preventing fluid evaporation
We extensively characterized the coordinated role of actin scaffolds and filaggrin granule formation during differentiation of normal human epidermal keratinocytes (NHEKs) (Gutowska-Owsiak et al, 2018)
We used a “calcium switch” model of keratinocyte differentiation, whereby differentiation is induced by addition of calcium (Bikle et al, 2012), reflecting physiological changes observed during epidermal stratification (Menon et al, 1985)
Summary
The epidermis, in the form of a stratified and desquamating tissue is a fascinating feature of many organisms; it retains very distinct characteristics which enable it to exert function, shielding us from external threats and preventing fluid evaporation. At the cellular level, the lamellar organization of lipids and their lateral packing properties prevent evaporation (Iwai et al, 2012; Janssens et al, 2012; Narangifard et al, 2018), forming a seal over the moisture trapped in the stratum corneum. This phenomenon is achieved by high abundance of hydrophilic compounds, known as Natural Moisturizing Factor (NMF)
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