Keratin (K) proteins are intermediate filament proteins that form the keratin cytoskeleton in keratinocytes, providing mechanical stability to the epidermis of the skin. In basal epidermal cells, K14 and K5 are expressed and form heterodimers. Mutations in the K14 gene are associated with the development of epidermolysis bullosa simplex (EBS), a disorder characterized by increased skin fragility and blister formation. The R125P mutation in the K14 gene causes alterations in the organization of the keratin cytoskeleton and may also affect keratinocyte adhesion and the expression of adhesion proteins. The aim of this master’s thesis was to evaluate the effect of the R125P mutation and its proportion on keratinocyte adhesion, as well as on the expression and localization of adhesion proteins. We also aimed to evaluate the effect of the amount of mutant keratin 14 on the organization of the keratin cytoskeleton in keratinocytes. For this study, we used NEB-1 cell lines expressing either EGFP-K14 wild-type or EGFP-K14 R125P constructs at different proportions. The expression of selected adhesion proteins was evaluated using SDS-PAGE followed by Western blotting, while their localization was assessed by immunofluorescence microscopy. Cell adhesion assays were performed to evaluate the ability of the cells to adhere to different substrates. Immunofluorescent labeling of K5 was used to assess the effect of the K14 mutation on the organization of the keratin cytoskeleton. Tissue models of skin were also prepared to analyze tissue organization and the expression of adhesion proteins. We found that different proportions of mutant K14 resulted in distinct changes in the expression and localization of adhesion proteins, as well as in the adhesive capacity of keratinocytes. The addition of an extracellular matrix component improved cell adhesion in the cell line containing 25% mutant K14, suggesting a potential rescue of the adhesion phenotype in this cell line. We also found that the organization of the keratin cytoskeleton varied depending on the amount of the expressed EGFP-K14 R125P construct.
|
