An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies.
The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further,...
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Public Library of Science (PLoS)
2024-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0300348&type=printable |
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| author | Collin M Douglas Jonathan E Bird Daniel Kopinke Karyn A Esser |
| author_facet | Collin M Douglas Jonathan E Bird Daniel Kopinke Karyn A Esser |
| author_sort | Collin M Douglas |
| collection | DOAJ |
| description | The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures. |
| format | Article |
| id | doaj-art-3a2aa2d6afbf429885d3f2d1c0687cfc |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-3a2aa2d6afbf429885d3f2d1c0687cfc2024-12-15T05:31:36ZengPublic Library of Science (PLoS)PLoS ONE1932-62032024-01-01194e030034810.1371/journal.pone.0300348An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies.Collin M DouglasJonathan E BirdDaniel KopinkeKaryn A EsserThe sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0300348&type=printable |
| spellingShingle | Collin M Douglas Jonathan E Bird Daniel Kopinke Karyn A Esser An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies. PLoS ONE |
| title | An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies. |
| title_full | An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies. |
| title_fullStr | An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies. |
| title_full_unstemmed | An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies. |
| title_short | An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies. |
| title_sort | optimized approach to study nanoscale sarcomere structure utilizing super resolution microscopy with nanobodies |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0300348&type=printable |
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