Each of these hypotheses is able to explain various BMS-599626 aspects of EBS pathophysiology and experimental data. Our recent work on the mechanical properties of intermediate filaments suggest that these filament networks are remarkably extensible, strong and tough, especially when compared to the other two cytoskeletal elements F-actin and microtubules. These findings are consistent with the fragile filament hypothesis, as disruptive mutations could have serious negative consequences for the material properties of individual keratin filaments. Russell et al. subjected an EBS keratinocyte cell line to cyclic mechanical stress and found that the keratin network collapses around the nucleus whereas WT networks do not. They also found evidence that the keratin network of these cells fragments into aggregate-like particles when mechanically stressed. On the surface, these results are consistent with the fragile filament hypothesis, but it is also possible that network breakdown was not caused directly by mechanical stress on the filaments, but rather by a generalized cellular stress response that then led to changes to the keratin network. In vitro investigations of K5/K14 filament suspensions are consistent with the fragile network hypothesis. These studies demonstrate that networks of filaments formed from EBS mutant keratin proteins are less stiff and less resilient than WT networks and appear to be deficient in their ability to form keratin bundles. Furthermore, the fact that EBS-like diseases can be caused by mutations in genes for IF cross-linking proteins like plectin suggest that IF-IF interactions are important for cell integrity and may contribute to the EBS phenotype. The sparse network hypothesis is consistent with observations that keratin filament densities are typically lower in EBS cells, especially those in which keratin proteins are tied up in aggregates. It is also supported by the fact that individuals homozygous for a K14 null mutation and conditional knockout mice lacking K5/K14 filaments both exhibit the EBS phenotype. In the current study, we focused on testing the fragile filament and fragile network hypotheses by subjecting keratinocytes expressing WT and EBS mutant K14-GFP to large scale uniaxial stretches. The GFP tags allowed us to monitor the morphology of the keratin network in live cells during stretch and test the prediction that keratin bundles and/or networks containing EBS mutant protein exhibit a defective response to being loaded in tension. We also investigated the possibility that the presence of mutant keratin proteins in EBS cells interferes with the other two cytoskeletal networks, F-actin and microtubules. We accomplished this by examining the response of the same cells to large-scale stretch in the presence and absence of F-actin and microtubule inhibitors. Here we demonstrate that K5/K14 filaments and networks containing mutant K14-R125P protein do not appear to be mechanically fragile or defective when subjected to large uniaxial cell strains. While the expression of mutant K14-GFP proteins in keratinocytes induced Pancuronium dibromide aggregates similar to those found in EBSDM cells, we found that the expression of these mutant proteins had no negative effects on keratinocyte viability after large-scale stretch. We also found that the presence of mutant protein had no effect on the response of the F-actin and microtubule networks to large-scale stretches.