title: The mechanical behavior of mutant K14-R125P keratin bundles and networks in NEB-1 keratinocytes.
abstract: Epidermolysis bullosa simplex (EBS) is an inherited skin-blistering disease that is caused by dominant mutations in the genes for keratin K5 or K14 proteins. While the link between keratin mutations and keratinocyte fragility in EBS patients is clear, the exact biophysical mechanisms underlying cell fragility are not known. In this study, we tested the hypotheses that mutant K14-R125P filaments and/or networks in human keratinocytes are mechanically defective in their response to large-scale deformations. We found that mutant filaments and networks exhibit no obvious defects when subjected to large uniaxial strains and have no negative effects on the ability of human keratinocytes to survive large strains. We also found that the expression of mutant K14-R125P protein has no effect on the morphology of the F-actin or microtubule networks or their responses to large strains. Disassembly of the F-actin network with Latrunculin A unexpectedly led to a marked decrease in stretch-induced necrosis in both WT and mutant cells. Overall, our results contradict the hypotheses that EBS mutant keratin filaments and/or networks are mechanically defective. We suggest that future studies should test the alternative hypothesis that keratinocytes in EBS cells are fragile because they possess a sparser keratin network.
authors.affiliation: Department of Integrative Biology, University of Guelph, Guelph, Canada.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Beriault
authors.forename: Oualid
authors.initials: O
authors.lastname: Haddad
authors.forename: John V
authors.initials: JV
authors.lastname: McCuaig
authors.forename: Zachary J
authors.initials: ZJ
authors.lastname: Robinson
authors.forename: David
authors.initials: D
authors.lastname: Russell
authors.forename: E Birgitte
authors.initials: EB
authors.lastname: Lane
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Fudge
mesh_terms: D000199:Actins / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D001696:Biomechanical Phenomena; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D003599:Cytoskeleton / Q000378:metabolism*; D016110:Epidermolysis Bullosa Simplex / Q000473:pathology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D053547:Keratin-14 / Q000737:chemistry / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism* / Q000473:pathology*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009997:Osmotic Pressure; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013314:Stress, Mechanical
chemical_list: D000199:Actins; C507540:KRT14 protein, human; D053547:Keratin-14; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins