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  1. Evaluation Summary:

    Kinetochores are large protein complexes that mediate faithful chromosome segregation in eukaryotes. The authors develop an in vitro system to study interactions between kinetochores and microtubules in yeast cell extracts and detect a role for a kinesin motor protein in the generation of kinetochore movements. This paper should be interesting to researchers working in the field of mitosis, molecular motors, the cell cycle, the cytoskeleton, and, more broadly, for those studying macromolecular complexes with reconstitution and in vitro imaging approaches.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript, the authors use a budding yeast cell lysate-based reconstitution system to investigate the movement of intact kinetochore particles along microtubules polymerized from yeast tubulin. Using TIRF microscopy, the authors directly observe kinetochore particles moving along microtubules toward the plus ends. After arriving to the tips, the microtubules typically undergo depolymerization, and the authors report the majority of kinetochore particles track along with the depolymerizing ends. The authors go on to determine that the plus-end activity driving the initial movement is due to the kinesin-8 motor Kip3, and the activity is unique to this plus-end motor. They also demonstrate that Kip3's motor activity, but not its tail domain, which promotes microtubule depolymerization, is necessary for processive plus end-directed kinetochore particle movement. To my knowledge, this is the first direct observation of single kinetochore particles undergoing lateral-to-end on conversion in a reconstituted in vitro system. In addition, the authors use their lysate-based approach to characterize a new role for the budding yeast kinesin-8 Kip3 in powering lateral kinetochore movement along microtubules.

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  3. Reviewer #2 (Public Review):

    The authors present microscopy data from observations of cell free yeast extracts demonstrating that kinetochores stably bind and translocate along the lattice of dynamic microtubules. This system is used to identify the process by which kinetochores convert from lateral association with microtubules to end-on attachment, a process that is difficult to study in vivo. Kinetochores in these experiments are labeled for imaging via fluorescent protein tags on endogenous kinetochore proteins. This provides the advantage of maintaining the endogenous stoichiometry of kinetochore proteins and their regulators in the in vitro imaging experiments. TIRF imaging of extracts from yeast with two different kinetochore complexes labeled, as well as correlative light and cryo-electron tomography experiments convincingly demonstrate that the kinetochores studied in this system are intact and contain all major subcomplexes. The directional movement of kinetochores described in this study has not been previously observed in vitro and could be an important mechanism contributing to the end-on attachment of kinetochores in vivo. Using extracts from yeast strains containing null mutations in the five kinesin genes found in budding yeast, the authors show that the directional movement depends, in large part, on the kinesin-8 Kip3. Studies of a Kip3 mutant deficient in promoting microtubule depolymerization suggest that this activity of Kip3 is also required for efficient end-on attachments. The authors provide an explanation for how these effects of Kip3 on kinetochores in the cell free extract could explain the unclustered kinetochore phenotype observed in Kip3 mutant mitotic cells, which has been a point of confusion in the field. The presented data highlight a potential new role for Kip3 in promoting end-on kinetochore attachments that could be relevant to understanding kinetochore microtubule attachment defects caused by loss of kinesin-8 function in other organisms, including Drosophila and mammals. Furthermore, the establishment of the cell free yeast extract system for mechanistic studies of kinetochore function is an important contribution.

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  4. Reviewer #3 (Public Review):

    The authors use a yeast extract system coupled with in-vitro imaging by TIRF microscopy, to study encounters between kinetochores and microtubules in metaphase-arrested yeast extracts. Using fluorescent labels on different subunits spanning the kinetochore, they observe lateral binding to microtubules, while a sizeable fraction of kinetochores displays plus-end directed movement. Upon reaching the plus-end kinetochores remain attached to depolymerizing microtubules. Dual color labeling and Cryo-ET suggest that entire kinetochrores are observed in this situation. In extracts lacking the kinesin-8 Kip3, plus-end directed movement is almost entirely absent, while addition of Kip3 containing extracts restores plus-end movement.

    Taken together, the development of an assay system to study kinetochore motility outside the context of a cell is an important step forward. The paper combines yeast extracts and the ability to use mutants and cell cycle arrests in an elegant manner and contains some interesting observations.

    There are some limitations of the study: The Kip3 deletion phenotype suggests that this motor only has a relatively modest contribution to end-on attachment and bi-orientation in cells. The molecular basis for Kip3 dependent movement of kinetochores in the extract remains somewhat unclear. There are some further conceptual limitations to the extracts used. For example, while there are nuclear and cytoplasmic fractions of Kip3 in yeast cells, in the whole cell extracts used here this compartmentalization of the cell is lost which can complicate the interpretation of results obtained in this system.

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