Keratins (K) are the largest group of intermediate filaments. They have a characteristic structure that causes them to organize into coiled dimers. K5 forms heterodimers with K14. They are mostly present in basal keratinocytes in the epidermis and their primary role is the physical stability of the epidermis. Mutations in K5 or K14 can cause epidermolysis bullosa simplex (EBS), which is characterized by skin fragility that results in blisters and erosions by minor mechanical trauma. In this master's thesis, we were interested in how the mutations E475G in K5, which causes severe EBS, and P25L in K5, which causes EBS with mottled pigmentation, effect the phenotype of keratinocytes grown in cell culture. We used the cell line NEB-1 with previously inserted construct EGFP-K5 wild type, E475G or P25L. Our goal was to select clones that express the construct in specific percentage in comparison with endogenous K5 and compare them. We used SDS-PAGE and Western blot to compare the quantity of EGFP-K5 and endogenous K5. We only got clones with the same, low, quantity of the construct. We used optical tweezers to measure the cortical stiffness of the cells and concluded that neither of the constructs causes important changes in the cortical stiffness and there are no bigger differences between the cell lines. The construct EGFP-K5 E475G, even in small amounts, increases spontaneous aggregate formation, where the aggregates are not only in the cell periphery. This is contrary to our predictions. Previous studies on cells with E475G mutation did not confirm that the mutation causes such disturbance of keratin cytoskeleton, where the spontaneous aggregate formation would be increased. We also noticed a higher expression of K16 and K19 in cells EGFP-K5 E475G. We created skin tissue models with keratinocytes expressing different constructs, where EGFP-K5 E475G stood out the most. We noticed several voids between the dermis and epidermis as well as between cells in epidermis, which is typical for severe EBS and shows the important role of keratin filaments in adhesion.
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