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.

Metrics

  • 584
    Page views
  • 90
    Downloads
  • 0
    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)

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

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

  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

Further reading

    1. Cell Biology
    2. Medicine
    Thao DV Le, Dianxin Liu ... Julio E Ayala
    Research Article Updated

    The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

    1. Cell Biology
    2. Developmental Biology
    Simon Schneider, Andjela Kovacevic ... Hubert Schorle
    Research Article

    Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.