The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

Abstract

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease CAPN7 (Calpain-7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem MIT domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for: 1) CAPN7 recruitment to midbodies, 2) efficient abscission, and 3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.

Data availability

X-Ray diffraction data were deposited in the PDB under accession code 8DFJ. NMR chemical shift assignments for IST1(303-366) are available at Biological Magnetic Resonance Bank (Accession no: 25393)(Caballe et al., 2015). All new plasmids generated for this study have been deposited at Addgene.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Elliott L Paine

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5575-297X
  2. Jack J Skalicky

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Frank G Whitby

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Douglas R Mackay

    Department of Oncological Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Katharine S Ullman

    Department of Oncological Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3693-2830
  6. Christopher P Hill

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6796-7740
  7. Wesley I Sundquist

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    For correspondence
    wes@biochem.utah.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9988-6021

Funding

National Institutes of Health (5R01GM112080)

  • Katharine S Ullman
  • Christopher P Hill
  • Wesley I Sundquist

National Institutes of Health (F31GM139318)

  • Elliott L Paine

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

Reviewing Editor

  1. M Joanne Lemieux, University of Alberta, Canada

Version history

  1. Preprint posted: October 18, 2022 (view preprint)
  2. Received: November 8, 2022
  3. Accepted: September 28, 2023
  4. Accepted Manuscript published: September 29, 2023 (version 1)
  5. Version of Record published: October 19, 2023 (version 2)

Copyright

© 2023, Paine 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. Elliott L Paine
  2. Jack J Skalicky
  3. Frank G Whitby
  4. Douglas R Mackay
  5. Katharine S Ullman
  6. Christopher P Hill
  7. Wesley I Sundquist
(2023)
The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission
eLife 12:e84515.
https://doi.org/10.7554/eLife.84515

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https://doi.org/10.7554/eLife.84515

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