Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits
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 mut...
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| Veröffentlicht in: | Molecular biology of the cell 2004-03, Vol.15 (3), p.990-1002 |
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| creator | Werner, Nicola Susann Windoffer, Reinhard Strnad, Pavel Grund, Christine Leube, Rudolf Eberhard Magin, Thomas Michael |
| description | 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 |
| doi_str_mv | 10.1091/mbc.E03-09-0687 |
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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. 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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.</description><subject>Actins - metabolism</subject><subject>Biological Transport - physiology</subject><subject>Cells, Cultured</subject><subject>Cytoskeleton - metabolism</subject><subject>Epidermolysis Bullosa Simplex - etiology</subject><subject>Epidermolysis Bullosa Simplex - genetics</subject><subject>Epidermolysis Bullosa Simplex - metabolism</subject><subject>Humans</subject><subject>Keratinocytes - cytology</subject><subject>Keratinocytes - metabolism</subject><subject>Keratins - genetics</subject><subject>Keratins - metabolism</subject><subject>Mutation - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>RNA, Small Interfering - metabolism</subject><issn>1059-1524</issn><issn>1939-4586</issn><issn>1059-1524</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1vFSEUhonR2FpduzOs3E0LFxiYpWmuH0kTN7omDJwbsQyMHMY4v8c_6kx7jUvPhnPC8z6bl5DXnF1zNvCbafTXRyY6NnSsN_oJueSDGDqpTP9025kaOq4O8oK8QPzOGJey18_JBZd9b6Q2l-T3cY4B6lTSihHpuKRU0FGM05zgV9fWGei0NNdiyUhdalBp-wY0rNlN0SMtp4f7HurGZOrXVvAeErSSqcuBVvgJLlFHfcmtxnHZTXvK-Z1v5SHeqss4l9r2n78uXMYlx4YvybOTSwivzu8V-fr--OX2Y3f3-cOn23d3nZeSt04JFRQYcdhGGy84c8Zo1Y-cjcE78EKeDqPSalAcTDCK98IIp5TyIXCtxBV5--ida_mxADY7RfSQkstQFrSaayaV_D_Ih16xg-k38OYR9LUgVjjZucbJ1dVyZvcC7VagBSYsG-xe4JZ4c1Yv4wThH39uTPwBP9ibCw</recordid><startdate>200403</startdate><enddate>200403</enddate><creator>Werner, Nicola Susann</creator><creator>Windoffer, Reinhard</creator><creator>Strnad, Pavel</creator><creator>Grund, Christine</creator><creator>Leube, Rudolf Eberhard</creator><creator>Magin, Thomas Michael</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>200403</creationdate><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</title><author>Werner, Nicola Susann ; Windoffer, Reinhard ; Strnad, Pavel ; Grund, Christine ; Leube, Rudolf Eberhard ; Magin, Thomas Michael</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c441t-535d5e83222278c310a88756b10bdcaec34f2b575951e8d8516383a555cdd1753</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Actins - metabolism</topic><topic>Biological Transport - physiology</topic><topic>Cells, Cultured</topic><topic>Cytoskeleton - metabolism</topic><topic>Epidermolysis Bullosa Simplex - etiology</topic><topic>Epidermolysis Bullosa Simplex - genetics</topic><topic>Epidermolysis Bullosa Simplex - metabolism</topic><topic>Humans</topic><topic>Keratinocytes - cytology</topic><topic>Keratinocytes - metabolism</topic><topic>Keratins - genetics</topic><topic>Keratins - metabolism</topic><topic>Mutation - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>RNA, Small Interfering - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Werner, Nicola Susann</creatorcontrib><creatorcontrib>Windoffer, Reinhard</creatorcontrib><creatorcontrib>Strnad, Pavel</creatorcontrib><creatorcontrib>Grund, Christine</creatorcontrib><creatorcontrib>Leube, Rudolf Eberhard</creatorcontrib><creatorcontrib>Magin, Thomas Michael</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular biology of the cell</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Werner, Nicola Susann</au><au>Windoffer, Reinhard</au><au>Strnad, Pavel</au><au>Grund, Christine</au><au>Leube, Rudolf Eberhard</au><au>Magin, Thomas Michael</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits</atitle><jtitle>Molecular biology of the cell</jtitle><addtitle>Mol Biol Cell</addtitle><date>2004-03</date><risdate>2004</risdate><volume>15</volume><issue>3</issue><spage>990</spage><epage>1002</epage><pages>990-1002</pages><issn>1059-1524</issn><eissn>1939-4586</eissn><eissn>1059-1524</eissn><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. 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| subjects | Actins - metabolism Biological Transport - physiology Cells, Cultured Cytoskeleton - metabolism Epidermolysis Bullosa Simplex - etiology Epidermolysis Bullosa Simplex - genetics Epidermolysis Bullosa Simplex - metabolism Humans Keratinocytes - cytology Keratinocytes - metabolism Keratins - genetics Keratins - metabolism Mutation - genetics Recombinant Proteins - metabolism RNA, Small Interfering - metabolism |
| 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 |
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