1. Cell Biology
  2. Physics of Living Systems

Three-dimensional structure of kinetochore-fibers in human mitotic spindles

  1. Robert Kiewisz  Is a corresponding author
  2. Gunar Fabig
  3. William Conway
  4. Daniel Baum
  5. Daniel J Needleman
  6. Thomas Müller-Reichert  Is a corresponding author
  1. Technische Universität Dresden, Germany
  2. Harvard University, United States
  3. Zuse Institute Berlin, Germany
https://doi.org/10.7554/eLife.75459
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Cite this article
  1. Robert Kiewisz
  2. Gunar Fabig
  3. William Conway
  4. Daniel Baum
  5. Daniel J Needleman
  6. Thomas Müller-Reichert
(2022)
Three-dimensional structure of kinetochore-fibers in human mitotic spindles
eLife 11:e75459.
https://doi.org/10.7554/eLife.75459
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Abstract

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.

Data availability

All Datasets were uploaded and ara available in OpaRA server:http://doi.org/10.25532/OPARA-128; http://doi.org/10.25532/OPARA-177he code used to perform quantitative analysis and visualization of MT organization in spindles has been uploaded to the GitHub repository and is available as open access under the GPL v3.0 license:https://github.com/RRobert92/ASGA; https://github.com/RRobert92/ASGA_3DViewer

The following data sets were generated

Article and author information

Author details

  1. Robert Kiewisz

    Experimental Center, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
    For correspondence
    robert.kiewisz@gmail.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2733-4978

  2. Gunar Fabig

    Experimental Center, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3017-0978

  3. William Conway

    Department of Physics, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7532-4331

  4. Daniel Baum

    Department of Visual and Data-Centric Computing, Zuse Institute Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.

  5. Daniel J Needleman

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Thomas Müller-Reichert

    Experimental Center, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
    For correspondence
    mueller-reichert@tu-dresden.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0203-1436

Funding

Deutsche Forschungsgemeinschaft (MU 1423/8-2)

  • Robert Kiewisz
  • Gunar Fabig
  • Thomas Müller-Reichert

Horizon 2020 Framework Programme (675737)

  • Robert Kiewisz
  • Thomas Müller-Reichert

Harvard University

  • William Conway
  • Daniel J Needleman

Nick Simons Foundation (1764269)

  • William Conway
  • Daniel J Needleman

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Adèle L Marston, University of Edinburgh, United Kingdom

Publication history

  1. Received: November 10, 2021
  2. Preprint posted: November 13, 2021 (view preprint)
  3. Accepted: July 24, 2022
  4. Accepted Manuscript published: July 27, 2022 (version 1)
  5. Version of Record published: August 10, 2022 (version 2)
  6. Version of Record updated: August 15, 2022 (version 3)

Copyright

© 2022, Kiewisz et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Robert Kiewisz
  2. Gunar Fabig
  3. William Conway
  4. Daniel Baum
  5. Daniel J Needleman
  6. Thomas Müller-Reichert
(2022)
Three-dimensional structure of kinetochore-fibers in human mitotic spindles
eLife 11:e75459.
https://doi.org/10.7554/eLife.75459

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Further reading

    1. Cell Biology
    2. Physics of Living Systems

    Self-organization of kinetochore-fibers in human mitotic spindles

    William Conway, Robert Kiewisz ... Daniel J Needleman
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    During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent reconstructions by electron tomography (Kiewisz et al., 2022) captured the positions and configurations of every MT in human mitotic spindles, revealing that roughly half the KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of large-scale electron tomography, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, with the speed of the KMT minus ends continually decreasing as the minus ends approach the pole, implying that longer KMTs grow more slowly than shorter KMTs. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores in metaphase and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

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