![]() During error correction, kinetochore–microtubule interactions are exchanged (swapped) if aberrant, but the exchange must stop when biorientation is established. To establish biorientation, aberrant kinetochore–microtubule interactions must be resolved through the error correction process. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.Ĭorrect chromosome segregation in mitosis relies on chromosome biorientation, in which sister kinetochores attach to microtubules from opposite spindle poles prior to segregation. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. ![]() In sum, our data show how a set of point mutations evolved in the histone-humanized yeasts to counterbalance human histone induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.ĭuring mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. ![]() Lastly, we show that one mutant, DAD1 E50D, while suppressing chromosome instability in mitosis, leads to gross defects in meiosis. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. Further, we characterize the molecular mechanism of two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Instead we show that a set of missense mutations in outer kinetochore proteins drive adaptation to human histones. Here we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes, defined by their centromeric evolutionary origins, however, they are not adaptive. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. The specific reduction of long-term stability relative to short-term strength might have important implications for mitotic error correction.įorcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. The third interaction region makes no apparent contribution to rupture strength, but its phosphorylation by Aurora B kinase specifically decreases the long-term stability of tip-coupling. ![]() The contribution of either of these two regions to tip-coupling is reduced by phosphorylation by Aurora B kinase. Here we show that interactions between two of these regions support the high rupture strengths that occur when applied force is rapidly increased and also support the stability of tip-coupling when force is held constant over longer durations. The functional relevance of these multiple interactions was mysterious. This tip-coupling behavior depends on the conserved Ndc80 complex and, in budding yeast, on the Dam1 complex, which bind each other directly via three distinct interacting regions. Accurate mitosis requires kinetochores to make persistent, load-bearing attachments to dynamic microtubule tips, thereby coupling chromosome movements to tip growth and shortening. ![]()
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