title: Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits.
abstract: Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.
authors.affiliation: Institut fuer Physiologische Chemie, Abteilung fuer Zellbiochemie, Universitaetsklinikum Bonn, 53115 Bonn, Germany.
authors.forename: Nicola Susann
authors.initials: NS
authors.lastname: Werner
authors.forename: Reinhard
authors.initials: R
authors.lastname: Windoffer
authors.forename: Pavel
authors.initials: P
authors.lastname: Strnad
authors.forename: Christine
authors.initials: C
authors.lastname: Grund
authors.forename: Rudolf Eberhard
authors.initials: RE
authors.lastname: Leube
authors.forename: Thomas Michael
authors.initials: TM
authors.lastname: Magin
mesh_terms: D000199:Actins / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D016110:Epidermolysis Bullosa Simplex / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D007633:Keratins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D007633:Keratins