Torvi, Wong et al. eLife 2022;0:e78450. DOI: https://doi.org/10.7554/eLife.78450 1 of 24 Reconstitution of kinetochore motility and microtubule dynamics reveals a role for a kinesin-8 in establishing end- on attachments Julia R Torvi 1,2† , Jonathan Wong 1† , Daniel Serwas 1 , Amir Moayed 1 , David G Drubin 1,2 , Georjana Barnes 1 * 1 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; 2 Biophysics Graduate Group, University of California, Berkeley, Berkeley, United States Abstract 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 fdelity chromosome capture and alignment at the mitotic midzone, but has been diffcult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal refection fuorescence (TIRF) microscopy to track the inter- actions 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 micro- tubule 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. Editor's evaluation Kinetochores are large protein complexes that mediate faithful chromosome segregation in eukary- otes. The authors develop an in vitro approach to study interactions between kinetochores and microtubules in yeast cell extracts. They use this powerful lysate-based system to characterize a new role for the budding yeast kinesin-8, Kip3, in powering lateral kinetochore movement along micro- tubules. This paper should be of interest to researchers working in the feld of mitosis, the cell cycle, and the cytoskeleton, and, more broadly, for those studying macromolecular complexes with recon- stitution and in vitro imaging approaches. Introduction During mitosis, a cell’s replicated chromosomes are equally segregated to two daughter cells. In order for this process to happen accurately and effciently, the microtubule cytoskeleton must completely remodel to form a bipolar spindle. Replicated chromosomes then attach to microtubules, emanating from opposite spindle poles, through a large dynamic chromosome-associated protein complex called RESEARCH ARTICLE *For correspondence: gbarnes@berkeley.edu † These authors contributed equally to this work Competing interest: The authors declare that no competing interests exist. Funding: See page 21 Preprinted: 08 March 2022 Received: 08 March 2022 Accepted: 21 June 2022 Published: 05 July 2022 Reviewing Editor: Jennifer G DeLuca, Colorado State University, United States Copyright Torvi, Wong et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.