During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. 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. 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. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. 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.

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. 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. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between endogenously tagged tubulin, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. Using both TIRF microscopy and cryo-correlative light microscopy and electron tomography, we successfully reconstituted microtubule-bound, intact kinetochores. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. 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.

TOG family proteins, including the budding yeast Stu2, are essential for the formation of a functional mitotic spindle. Across all eukaryotes, the described functions of this family depend on two microtubule binding elements: TOG domain arrays, and a basic linker domain important for binding the microtubule lattice. Consistently, we find here that Stu2's basic linker is required for its ability to regulate microtubules in vitro, including stimulating microtubule growth, shrinkage, and catastrophe. We furthermore define a region contained within Stu2's basic linker domain as its nuclear localization sequence, and identify phospho-regulation that promotes mitosis-specific nuclear import. Surprisingly, directing nuclear localization is the only function contained within Stu2's basic linker that is required for cell viability, indicating that microtubule lattice binding is not required for Stu2's essential function. Considering that lattice binding is required to stimulate microtubule polymerization and depolymerization in vitro, these established activities are unlikely to be the essential functions carried out by Stu2 in the cell's nucleus.

No abstract

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast,Saccharomyces cerevisiae. Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.