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
Share this article
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
  2. Download BibTeX
  3. Download .RIS

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.

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.

Metrics

  • 3,351
    views
  • 540
    downloads
  • 54
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

Share this article

https://doi.org/10.7554/eLife.75459

Categories and tags

Research organism

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
    Research Article Updated

    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.

    1. Cancer Biology
    2. Cell Biology

    Cytosolic and endoplasmic reticulum chaperones inhibit wt-p53 to increase cancer cells' survival by refluxing ER-proteins to the cytosol

    Salam Dabsan, Gali Zur ... Aeid Igbaria
    Research Article

    The endoplasmic reticulum (ER) is an essential sensing organelle responsible for the folding and secretion of almost one-third of eukaryotic cells' total proteins. However, environmental, chemical, and genetic insults often lead to protein misfolding in the ER, accumulating misfolded proteins, and causing ER stress. To solve this, several mechanisms were reported to relieve ER stress by decreasing the ER protein load. Recently, we reported a novel ER surveillance mechanism by which proteins from the secretory pathway are refluxed to the cytosol to relieve the ER of its content. The refluxed proteins gain new prosurvival functions in cancer cells, thereby increasing cancer cell fitness. We termed this phenomenon ER to CYtosol Signaling (or ‘ERCYS’). Here, we found that in mammalian cells, ERCYS is regulated by DNAJB12, DNAJB14, and the HSC70 cochaperone SGTA. Mechanistically, DNAJB12 and DNAJB14 bind HSC70 and SGTA - through their cytosolically localized J-domains to facilitate ER-protein reflux. DNAJB12 is necessary and sufficient to drive this phenomenon to increase AGR2 reflux and inhibit wt-p53 during ER stress. Mutations in DNAJB12/14 J-domain prevent the inhibitory interaction between AGR2-wt-p53. Thus, targeting the DNAJB12/14-HSC70/SGTA axis is a promising strategy to inhibit ERCYS and impair cancer cell fitness.

    1. Cancer Biology
    2. Cell Biology

    N6-methyladenosine in DNA promotes genome stability

    Brooke A Conti, Leo Novikov ... Mariano Oppikofer
    Research Article

    DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluorouracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), and unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, and these results were confirmed using an orthogonal approach, liquid chromatography coupled to tandem mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base-damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA in promoting genome stability in mammalian cells, especially in response to base damage.