1. Cell Biology
  2. Developmental Biology

Optogenetic dissection of mitotic spindle positioning in vivo

  1. Lars-Eric Fielmich
  2. Ruben Schmidt
  3. Daniel J Dickinson
  4. Bob Goldstein
  5. Anna Akhmanova
  6. Sander Van den Heuvel  Is a corresponding author
  1. Utrecht University, Netherlands
  2. University of North Carolina at Chapel Hill, United States
https://doi.org/10.7554/eLife.38198
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  1. Lars-Eric Fielmich
  2. Ruben Schmidt
  3. Daniel J Dickinson
  4. Bob Goldstein
  5. Anna Akhmanova
  6. Sander Van den Heuvel
(2018)
Optogenetic dissection of mitotic spindle positioning in vivo
eLife 7:e38198.
https://doi.org/10.7554/eLife.38198
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Abstract

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα-GDP, Gα-GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

Data availability

Our design algorithm is accessible via a web interface at http://104.131.81.59/, and the source code can be found at https://github.com/dannyhmg/germline. The data that support the findings of this study are included in the supplementary information. All key plasmids and strains will be deposited with Addgene and the CGC upon publication.

The following previously published data sets were used

Article and author information

Author details

  1. Lars-Eric Fielmich

    Department of Biology, Utrecht University, Utrecht, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0247-1298

  2. Ruben Schmidt

    Department of Biology, Utrecht University, Utrecht, Netherlands
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9187-5424

  3. Daniel J Dickinson

    Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.

  4. Bob Goldstein

    Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, United States
    Competing interests
    No competing interests declared.

  5. Anna Akhmanova

    Department of Biology, Utrecht University, Utrecht, Netherlands
    Competing interests
    Anna Akhmanova, Deputy editor of eLife Magazine.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9048-8614

  6. Sander Van den Heuvel

    Department of Biology, Utrecht University, Utrecht, Netherlands
    For correspondence
    s.j.l.vandenheuvel@uu.nl
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9015-7463

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO TOP/ECHO grant 711.015.001)

  • Lars-Eric Fielmich
  • Sander Van den Heuvel

National Institutes of Health (NIH R01 GM083071)

  • Bob Goldstein

Helen Hay Whitney Foundation

  • Daniel J Dickinson

European Research Council (Synergy grant 609822)

  • Ruben Schmidt
  • Anna Akhmanova

National Institutes of Health (NIH K99 GM115964)

  • Daniel J Dickinson

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

Copyright

© 2018, Fielmich 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. Lars-Eric Fielmich
  2. Ruben Schmidt
  3. Daniel J Dickinson
  4. Bob Goldstein
  5. Anna Akhmanova
  6. Sander Van den Heuvel
(2018)
Optogenetic dissection of mitotic spindle positioning in vivo
eLife 7:e38198.
https://doi.org/10.7554/eLife.38198

Share this article

https://doi.org/10.7554/eLife.38198

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Further reading

    1. Cell Biology

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

    Masako Okumura, Toyoaki Natsume ... Tomomi Kiyomitsu
    Research Article Updated

    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.