A 'hot-spot' mutation alters the mechanical properties of keratin filamentnetworks

Keratins 5 and 14 polymerize to form the intermediate filament network in t he progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disord er(1). The impact that such mutations exert on the intrinsic mechanical pro perties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatl y reduces the ability of reconstituted mutant filaments to bundle under cro sslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant kera tins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probe s the local organization of filament networks, shows that the mutant polyme r exhibits highly heterogeneous structures compared to those of wild-type f ilaments. Our results indicate that the fragility of epithelial cells expre ssing mutant keratin may result from an impaired ability of keratin polymer s to be crosslinked into a functional network.