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 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.
Department of Biochemistry, University of Washington, Seattle, WA 98195.
Kimberly K
KK
Fong
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
Krishna K
KK
Sarangapani
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
Erik C
EC
Yusko
Department of Biochemistry, University of Washington, Seattle, WA 98195.
Michael
M
Riffle
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195.
Aida
A
Llauró
Department of Biochemistry, University of Washington, Seattle, WA 98195.
Beth
B
Graczyk
Department of Biochemistry, University of Washington, Seattle, WA 98195.
Trisha N
TN
Davis
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195 casbury@uw.edu.
Charles L
CL
Asbury
D001696:Biomechanical Phenomena / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D018385:Centrosome / Q000378:metabolism* / Q000502:physiology; D020090:Chromosome Segregation; D018386:Kinetochores / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000502:physiology; D008938:Mitosis; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D064048:Spindle Pole Bodies / Q000502:physiology*
D002147:Calmodulin; D029701:Saccharomyces cerevisiae Proteins