Keratin filaments are a major structural element in epithelial cells. They maintain shape and stiffness of cells by providing physical resistance to mechanical stress. Several different mutations in keratins leading to different types of disease have been described. Mutations in keratins 5 and 14 are known to cause the formation of epidermolysis bullosa simplex (EBS), a disease of skin fragility. In the master's thesis, we studied the point mutation K14 R125P, which causes a severe form of EBS. A typical phenotypic phenomenon with mutation is the formation of aggregates, which has not yet been explained in detail. Using optical tweezers, the stiffness of keratinocytes was determined by inserting a construct with additional copy of K14, with the R125P mutation, for different amounts of mutated keratin. We found that the difference in stiffness occurs only in cells with the lowest amount of mutated keratin, where we also observed the most aggregates. Aggregates are a dynamic organelle that can be recycled by a cell through a proteosomal system, as demonstrated by inhibition and western transfer. With mathematical modeling we also explained the formation of aggregates - with asymmetric binding in which both wild-type keratin and mutated keratin participate, and later the aggregates grow with the addition of mainly mutated keratin.
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