1. Evaluation Summary:

    Kiewisz and colleagues performed sophisticated reconstructions of kinetochore-fibers within human spindles using electron tomography, and then analyzed the ultrastructure and organization of their microtubules. This work will not only serve as an incredible resource for the field, but has clear implications for models of kinetochore-fiber and spindle self-organization.

    (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 #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    This paper is a technical tour de force, as demonstrated best in the many videos associated with the text. They reveal the huge amount of microtubule tracking that has been achieved and show HeLa spindles with more clarity and detail than has previously been accomplished. Thus, the paper is a landmark in spindle study. In its current form, however, the paper contains some technical errors and some issues with interpretations, and their remedy would make this paper an important classic in structural cell biology. These issues include: taking account of the collapse in section thickness that is brought about by the electron beam; recognizing that the great number of non-KMTs near the pole will bias the probability of MT-MT interactions in ways that should be taken into account; and re-examining the data to see if additional issues, such as the opened or closed status of KMTs at their polar ends can be determined. With these and other improvements, the paper will become a classic in the field.

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  3. Reviewer #2 (Public Review):

    In their manuscript, "Three-dimensional structure of kinetochore-fibers in human mitotic spindles," Kiewisz and colleagues performed sophisticated reconstructions of human kinetochore-fibers using electron tomography, and then analyzed the ultrastructure and organization of their kinetochore-microtubules. Previous work has been done to analyze k-fiber structures by EM in other cell types and species, but this manuscript represents the most comprehensive reconstruction of k-fibers to date across three human spindles. Here, the authors determine the number, length and morphology of KMTs, as well as their positioning and interactions relative to the MT network, revealing key differences in the makeup and organization of KMTs versus non-KMTs.

    We appreciate the rigorous experimental design and analysis, and the pertinence of choosing to work on k-fiber ultrastructure. The experimental logic is clear, the analysis is methodical, and the data are presented transparently and clearly. Overall, this work will be a great asset to the scientific community, though the main point of the paper and of certain figures requires more motivation and context.

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  4. Reviewer #3 (Public Review):

    Kiewisz et al present analysis of (traced) microtubules in the spindles generated from 3D tomography data for 3 human cells. As a full complement of MTs in human spindles this is a phenomenally rich data set and a fantastic reference resource. The authors' analysis provides crucial quantification of the human spindle and new insight into spindle architecture, and essential numbers for modellers. A key result is that bundles of KMTs starting at the kinetochore deteriorate through KMT termination and splay in going to the pole. They have made available a visualisation tool so readers can examine the data themselves - this is particularly welcome and aids understanding of their results.

    The authors discriminate kinetochore MTs (ending at KTs) and non-KMTs, counting both sets. They restrict analysis predominantly to the KMTs, quantifying number per KT, length, tortuosity (a proxi for curvature), 'bundle' deterioration from KT to pole, minus end proximity to other MTs and length-wise associations. The data appears consistent with previous studies eg O'Toole et al, 2020 but based on substantially higher samples of kinetochores/K-fibers (complete cell MT complement); one key new result is the heterogeneity and stochasticity in bundles of KMTs. Only 50% of KMTs reach the spindle pole zone and the (KMT) bundles are sometimes compact along their length or are only compact at the KT with significant splay (and KMT curvature) moving towards the pole. The KMTs in the periphery of the metaphase plate are less likely to reach the pole compared to those near the centre of the metaphase plate. There are thus substantial numbers of minus ends in the spindle, in fact from both KMTs and non-KMTs. An analysis of KMT proximity to other MTs is then presented - this suggests that KMT interactions increase in the pole, probably unsurprising as MT density increases near the poles (although allowance for this effect is not discussed).

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