1. Cell Biology
Download icon

Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

  1. Masako Okumura
  2. Toyoaki Natsume
  3. Masato T Kanemaki
  4. Tomomi Kiyomitsu  Is a corresponding author
  1. Nagoya University, Japan
  2. National Institute of Genetics, Japan
Research Article
  • Cited 47
  • Views 5,157
  • Annotations
Cite this article as: eLife 2018;7:e36559 doi: 10.7554/eLife.36559

Abstract

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

Data availability

All data generated or analyzed during this study are included in the manuscript, figures and supplemental files. We will deposit all plasmids and cell lines used in this study to non-profit organization such as Addgene and RIKEN BioResource Research Center.

Article and author information

Author details

  1. Masako Okumura

    Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
    Competing interests
    The authors declare that no competing interests exist.
  2. Toyoaki Natsume

    Division of Molecular Cell Engineering, National Institute of Genetics, Mishima, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3544-4491
  3. Masato T Kanemaki

    Division of Molecular Cell Engineering, National Institute of Genetics, Mishima, Japan
    Competing interests
    The authors declare that no competing interests exist.
  4. Tomomi Kiyomitsu

    Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
    For correspondence
    kiyomitsu@bio.nagoya-u.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2280-4611

Funding

Japan Science and Technology Agency (JPMJPR13A3)

  • Tomomi Kiyomitsu

Human Frontier Science Program (CDA00057/2014-C)

  • Tomomi Kiyomitsu

Japan Society for the Promotion of Science (16K14721)

  • Tomomi Kiyomitsu

Uehara Memorial Foundation

  • Tomomi Kiyomitsu

Naito Foundation

  • Tomomi Kiyomitsu

Japan Society for the Promotion of Science (17H05002)

  • Tomomi Kiyomitsu

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

Reviewing Editor

  1. Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: March 10, 2018
  2. Accepted: May 26, 2018
  3. Accepted Manuscript published: May 31, 2018 (version 1)
  4. Version of Record published: July 9, 2018 (version 2)

Copyright

© 2018, Okumura 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

  • 5,157
    Page views
  • 903
    Downloads
  • 47
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cell Biology
    Christina L Hueschen et al.
    Research Article

    To build the spindle at mitosis, motors exert spatially regulated forces on microtubules. We know that dynein pulls on mammalian spindle microtubule minus-ends, and this localized activity at ends is predicted to allow dynein to cluster microtubules into poles. How dynein becomes enriched at minus-ends is not known. Here, we use quantitative imaging and laser ablation to show that NuMA targets dynactin to minus-ends, localizing dynein activity there. NuMA is recruited to new minus-ends independently of dynein and more quickly than dynactin; both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves a C-terminal region outside NuMA’s canonical microtubule-binding domain and is independent of minus-end binders γ-TuRC, CAMSAP1, and KANSL1/3. Both NuMA’s minus-end-binding and dynein-dynactin-binding modules are required to rescue focused, bipolar spindle organization. Thus, NuMA may serve as a mitosis-specific minus-end cargo adaptor, targeting dynein activity to minus-ends to cluster spindle microtubules into poles.

    1. Cell Biology
    Andrea Serra-Marques, Sophie Dumont
    Insight

    Optogenetic approaches are leading to a better understanding of the forces that determine the plane of cell division.