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Direct measurement of the strength of microtubule attachment to yeast centrosomes

机译:直接测量微管与酵母中心体的附着强度

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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.
机译:酵母中的中心体或纺锤体极体(SPB)是至关重要的机械集线器,在有丝分裂纺锤体装配,纺锤体定位和染色体分离过程中,它们可保持微管的承重附件。然而,微管-中心体附着的强度是未知的,并且几乎没有研究过机械力可能调节中心体功能的可能性。为了揭示中心体如何维持和调节力,我们从发芽的酵母中纯化了SPB,并使用激光捕获在体外操纵了单个附着的微管。我们的实验表明,SPB-微管附件非常坚固,在相同的载荷条件下,其断裂力比动线体附件高大约四倍。此外,从SPB组件Spc110中除去钙调蛋白结合位点会在体外削弱SPB-微管的附着,并使细胞对体内SPB应激的增加敏感。这些观察结果表明钙调蛋白结合有助于SPB的机械完整性,并表明当主轴力升高时,其去除可能导致极分层和有丝分裂失败。我们建议非常高强度的SPB-微管附件对于有丝分裂细胞中的纺锤体完整性可能很重要,这样在动植物中产生的拉力不会引起SPB上的微管分离和分层。

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