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Direct measurement of the strength of microtubule attachment to yeast centrosomes

Centrosomes, or spindle pole bodies (SPBs) in yeast, are vital mechanical hubs that maintain load-bearing attachments to microtubules during mitotic spindle assembly, spindle positioning, and chromosome segregation. However, the strength of microtubule-centrosome attachments is unknown, and the poss...

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Autores principales: Fong, Kimberly K., Sarangapani, Krishna K., Yusko, Erik C., Riffle, Michael, Llauró, Aida, Graczyk, Beth, Davis, Trisha N., Asbury, Charles L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541836/
https://www.ncbi.nlm.nih.gov/pubmed/28331072
http://dx.doi.org/10.1091/mbc.E17-01-0034
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author Fong, Kimberly K.
Sarangapani, Krishna K.
Yusko, Erik C.
Riffle, Michael
Llauró, Aida
Graczyk, Beth
Davis, Trisha N.
Asbury, Charles L.
author_facet Fong, Kimberly K.
Sarangapani, Krishna K.
Yusko, Erik C.
Riffle, Michael
Llauró, Aida
Graczyk, Beth
Davis, Trisha N.
Asbury, Charles L.
author_sort Fong, Kimberly K.
collection PubMed
description Centrosomes, or spindle pole bodies (SPBs) in yeast, are vital mechanical hubs that maintain load-bearing attachments to microtubules during mitotic spindle assembly, spindle positioning, and chromosome segregation. However, the strength of microtubule-centrosome attachments is unknown, and the possibility that mechanical force might regulate centrosome function has scarcely been explored. To uncover how centrosomes sustain and regulate force, we purified SPBs from budding yeast and used laser trapping to manipulate single attached microtubules in vitro. Our experiments reveal that SPB–microtubule attachments are extraordinarily strong, rupturing at forces approximately fourfold higher than kinetochore attachments under identical loading conditions. Furthermore, removal of the calmodulin-binding site from the SPB component Spc110 weakens SPB–microtubule attachment in vitro and sensitizes cells to increased SPB stress in vivo. These observations show that calmodulin binding contributes to SPB mechanical integrity and suggest that its removal may cause pole delamination and mitotic failure when spindle forces are elevated. We propose that the very high strength of SPB–microtubule attachments may be important for spindle integrity in mitotic cells so that tensile forces generated at kinetochores do not cause microtubule detachment and delamination at SPBs.
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spelling pubmed-55418362017-09-22 Direct measurement of the strength of microtubule attachment to yeast centrosomes Fong, Kimberly K. Sarangapani, Krishna K. Yusko, Erik C. Riffle, Michael Llauró, Aida Graczyk, Beth Davis, Trisha N. Asbury, Charles L. Mol Biol Cell Brief Reports Centrosomes, or spindle pole bodies (SPBs) in yeast, are vital mechanical hubs that maintain load-bearing attachments to microtubules during mitotic spindle assembly, spindle positioning, and chromosome segregation. However, the strength of microtubule-centrosome attachments is unknown, and the possibility that mechanical force might regulate centrosome function has scarcely been explored. To uncover how centrosomes sustain and regulate force, we purified SPBs from budding yeast and used laser trapping to manipulate single attached microtubules in vitro. Our experiments reveal that SPB–microtubule attachments are extraordinarily strong, rupturing at forces approximately fourfold higher than kinetochore attachments under identical loading conditions. Furthermore, removal of the calmodulin-binding site from the SPB component Spc110 weakens SPB–microtubule attachment in vitro and sensitizes cells to increased SPB stress in vivo. These observations show that calmodulin binding contributes to SPB mechanical integrity and suggest that its removal may cause pole delamination and mitotic failure when spindle forces are elevated. We propose that the very high strength of SPB–microtubule attachments may be important for spindle integrity in mitotic cells so that tensile forces generated at kinetochores do not cause microtubule detachment and delamination at SPBs. The American Society for Cell Biology 2017-07-07 /pmc/articles/PMC5541836/ /pubmed/28331072 http://dx.doi.org/10.1091/mbc.E17-01-0034 Text en © 2017 Fong et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
spellingShingle Brief Reports
Fong, Kimberly K.
Sarangapani, Krishna K.
Yusko, Erik C.
Riffle, Michael
Llauró, Aida
Graczyk, Beth
Davis, Trisha N.
Asbury, Charles L.
Direct measurement of the strength of microtubule attachment to yeast centrosomes
title Direct measurement of the strength of microtubule attachment to yeast centrosomes
title_full Direct measurement of the strength of microtubule attachment to yeast centrosomes
title_fullStr Direct measurement of the strength of microtubule attachment to yeast centrosomes
title_full_unstemmed Direct measurement of the strength of microtubule attachment to yeast centrosomes
title_short Direct measurement of the strength of microtubule attachment to yeast centrosomes
title_sort direct measurement of the strength of microtubule attachment to yeast centrosomes
topic Brief Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541836/
https://www.ncbi.nlm.nih.gov/pubmed/28331072
http://dx.doi.org/10.1091/mbc.E17-01-0034
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