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A more recent version of this article appeared on March 1, 2004
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Submitted on September 24, 2003
Revised on October 20, 2003
Accepted on October 31, 2003
1 Institut fuer Physiologische Chemie, Abteilung fuer Zellbiochemie, Universitaetsklinikum Bonn, Nussallee 11, D-53115 Bonn, Germany
2 Department of Anatomy, Johannes Gutenberg-University, Becherweg 13, D-55128 Mainz, Germany
3 Division of Cell Biology, German Cancer Research Center, Heidelberg
* Corresponding author. E-mail address: t.magin{at}uni-bonn.de.
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 EYFP-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 wildtype protein. In support, K14R125C-specific RNAi 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 EBS using siRNAs.
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