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Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin
To learn how nebulin functions in the assembly and maintenance of I-Z-I bands, MYC- and GFP- tagged nebulin fragments were expressed in primary cultured skeletal myotubes. Their sites of incorporation were visualized by double staining with anti-MYC, antibodies to myofibrillar proteins, and FITC- or...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2000
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175182/ https://www.ncbi.nlm.nih.gov/pubmed/10931867 |
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author | Ojima, K. Lin, Z.X. Bang, M.-L. Holtzer, S. Matsuda, R. Labeit, S. Sweeney, H.L. Holtzer, H. |
author_facet | Ojima, K. Lin, Z.X. Bang, M.-L. Holtzer, S. Matsuda, R. Labeit, S. Sweeney, H.L. Holtzer, H. |
author_sort | Ojima, K. |
collection | PubMed |
description | To learn how nebulin functions in the assembly and maintenance of I-Z-I bands, MYC- and GFP- tagged nebulin fragments were expressed in primary cultured skeletal myotubes. Their sites of incorporation were visualized by double staining with anti-MYC, antibodies to myofibrillar proteins, and FITC- or rhodamine phalloidin. Contrary to expectations based on in vitro binding studies, none of the nebulin fragments expressed in maturing myotubes were incorporated selectively into I-band ∼1.0-μm F-α-actin–containing thin filaments. Four of the MYC/COOH-terminal nebulin fragments were incorporated exclusively into periodic ∼0.1-μm Z-bands. Whereas both anti-MYC and Rho-phalloidin stained intra-Z-band F-α-actin oligomers, only the latter stained the pointed ends of the polarized ∼1.0-μm thin filaments. Z-band incorporation was independent of the nebulin COOH-terminal Ser or SH3 domains. In vitro cosedimentation studies also demonstrated that nebulin SH3 fragments did not bind to F-α-actin or α-actinin. The remaining six fragments were not incorporated into Z-bands, but were incorporated (a) diffusely throughout the sarcoplasm and into (b) fibrils/patches of varying lengths and widths nested among normal striated myofibrils. Over time, presumably in response to the mediation of muscle-specific homeostatic controls, many of the ectopic MYC-positive structures were resorbed. None of the tagged nebulin fragments behaved as dominant negatives; they neither blocked the assembly nor induced the disassembly of mature striated myofibrils. Moreover, they were not cytotoxic in myotubes, as they were in the fibroblasts and presumptive myoblasts in the same cultures. |
format | Text |
id | pubmed-2175182 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2000 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21751822008-05-01 Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin Ojima, K. Lin, Z.X. Bang, M.-L. Holtzer, S. Matsuda, R. Labeit, S. Sweeney, H.L. Holtzer, H. J Cell Biol Original Article To learn how nebulin functions in the assembly and maintenance of I-Z-I bands, MYC- and GFP- tagged nebulin fragments were expressed in primary cultured skeletal myotubes. Their sites of incorporation were visualized by double staining with anti-MYC, antibodies to myofibrillar proteins, and FITC- or rhodamine phalloidin. Contrary to expectations based on in vitro binding studies, none of the nebulin fragments expressed in maturing myotubes were incorporated selectively into I-band ∼1.0-μm F-α-actin–containing thin filaments. Four of the MYC/COOH-terminal nebulin fragments were incorporated exclusively into periodic ∼0.1-μm Z-bands. Whereas both anti-MYC and Rho-phalloidin stained intra-Z-band F-α-actin oligomers, only the latter stained the pointed ends of the polarized ∼1.0-μm thin filaments. Z-band incorporation was independent of the nebulin COOH-terminal Ser or SH3 domains. In vitro cosedimentation studies also demonstrated that nebulin SH3 fragments did not bind to F-α-actin or α-actinin. The remaining six fragments were not incorporated into Z-bands, but were incorporated (a) diffusely throughout the sarcoplasm and into (b) fibrils/patches of varying lengths and widths nested among normal striated myofibrils. Over time, presumably in response to the mediation of muscle-specific homeostatic controls, many of the ectopic MYC-positive structures were resorbed. None of the tagged nebulin fragments behaved as dominant negatives; they neither blocked the assembly nor induced the disassembly of mature striated myofibrils. Moreover, they were not cytotoxic in myotubes, as they were in the fibroblasts and presumptive myoblasts in the same cultures. The Rockefeller University Press 2000-08-07 /pmc/articles/PMC2175182/ /pubmed/10931867 Text en © 2000 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Ojima, K. Lin, Z.X. Bang, M.-L. Holtzer, S. Matsuda, R. Labeit, S. Sweeney, H.L. Holtzer, H. Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin |
title | Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin |
title_full | Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin |
title_fullStr | Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin |
title_full_unstemmed | Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin |
title_short | Distinct Families of Z-Line Targeting Modules in the Cooh-Terminal Region of Nebulin |
title_sort | distinct families of z-line targeting modules in the cooh-terminal region of nebulin |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175182/ https://www.ncbi.nlm.nih.gov/pubmed/10931867 |
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