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  1. Evaluation Summary:

    Conway and colleagues use a combination of experiments and theory to test models for how kinetochore-fibers are born in mammalian spindles. Their work is consistent with a model where kinetochore-fibers primarily nucleate de novo at kinetochores, rather than arise from search-and-capture of microtubules. This work should be of interest to experimentalists and theorists broadly interested in self-organization, and in cell division.

    (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 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Conway et al. use high quality light microscopy to observe microtubule (MT) arrangements and dynamics in live HeLa cells at metaphase. Local photoactivation of fluorescence produces a bar of labeled tubulin that migrates poleward, as shown previously by several groups on several types of cells. In this system, the intensity of the bar (corrected for photobleaching) decreases at a rate best fit by two exponentials, apparently corresponding to the fast turnover of non-kinetochore MTs and the slower turnover of the kinetochore MTs (KMTs) themselves. Moreover, the bar spreads significantly as it migrates poleward. The speed of the bar's peak along the spindle axis slows as it approaches the pole and is less for bars generated near the spindle pole. These results are interpreted to mean that KMTs flux poleward at different rates in different parts of the spindle. This slowing is used to evaluate models for KMT turnover in the HeLa spindle, whose structure is modeled with the concept of an active liquid crystal. The fraction of MT fluorescence that turns over slowly corresponds nicely with the fraction of MTs near the spindle equator that ends on kinetochores, as determined in a companion paper that uses electron tomography, so slowly fading fluorescence is interpreted as a metric for KMTs. This leads the authors to conclude that there are no slowly turning over MTs other than KMTs. The authors then compare spindle structure as seen by high quality polarization microscopy and by electron tomography with a computational model.

  3. Reviewer #2 (Public Review):

    In their manuscript, "Self-organization of kinetochore-fibers in human mitotic spindles," Conway and colleagues use a combination of photomarking, electron tomography, polarized light microscopy and theory to test models for how they kinetochore-fibers are born in the mammalian spindle. Indeed, how KMTs in mammalian spindles self-organize to build a k-fiber remains poorly understood. This work addresses this gap and contributes two major advances. First, while it is known that microtubule flux can vary regionally in spindles of different architectures in other species, the authors report on spatial flux differences in human spindles, which has implications for how the mammalian spindle builds and maintains itself. Second, the authors develop a model that describes KMT self-organization in human spindles and combine it with their experimental data to test models for how k-fibers are born. This model supports nucleation of KMTs predominantly at kinetochores as opposed to a search-and-capture model.

    We appreciate the pertinence of investigating mammalian k-fibers, the new and careful experimental data, as well as the model the authors have developed to describe k-fiber self-organization. This work will be an important contribution to the field, both for the framework it provides and for the conclusion it makes on how k-fibers are born, though it could be improved for clarity and accessibility.

  4. Reviewer #3 (Public Review):

    In the present work, Conway et al. investigate the origin of kinetochore microtubules in human HeLa cells by combining electron tomography data showing that about 50% of kinetochore microtubule minus ends do not end at the kinetochore, with measurements of microtubule dynamic properties in living cells showing that kinetochore microtubules grow away from kinetochores along defined trajectories and slowing down near the poles, as well as theoretical modeling to test models of kinetochore microtubule origin for comparison with experimental data from cells. The authors find that the fraction of kinetochore microtubules directly determined by their electron-tomographic reconstructions matches to perfection the slow turnover component of a double exponential fit obtained from photoconversion of fluorescent tubulin in living cells. To my knowledge, this constitutes the most definitive demonstration of a long-proposed concept that this slow turnover population corresponds essentially to the entire kinetochore microtubules in the mitotic spindle. This is timely, as a recent work from the Gorbsky lab suggested the existence of more than two microtubule populations in the spindle. Availability of the present data, will allow the field to properly evaluate the significance of the findings and respective interpretations. Another interesting aspect provided by the present manuscript is the finding that flux in human cells decreases speed near the spindle poles, but kinetochore (and non-kinetochore) microtubule turnover was uniform along the spindle. Pol-scope analysis (and theory) revealed that both microtubule populations were highly aligned. Last, the authors use biophysical modeling to confront two possible models of kinetochore microtubule origin based on many of the parameters measured experimentally. The results show that a "kinetochore nucleation" model best explains the experimental results obtained from living cells. Overall, the authors propose an integrated model that predicts the lengths, orientations, and dynamics of kinetochore microtubules in mitotic spindles of human HeLa cells. Although many of the proposed concepts are not novel per se and have been demonstrated in other systems, including metazoans, the validation in human cell models is really the highest value of the present work. The writing of the manuscript is rigorous and the interpretations carefully discussed and well supported by the data and theory. This work will certainly change the way we conceive spindle assembly and kinetochore-microtubule formation in mammalian cells, and will motivate a revision of current textbooks.