The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding

  1. Tatyana Bodrug
  2. Elizabeth M Wilson-Kubalek
  3. Stanley Nithianantham
  4. Alex F Thompson
  5. April Alfieri
  6. Ignas Gaska
  7. Jennifer Major
  8. Garrett Debs
  9. Sayaka Inagaki
  10. Pedro Gutierrez
  11. Larisa Gheber
  12. Richard J McKenney
  13. Charles Vaughn Sindelar
  14. Ron Milligan
  15. Jason Stumpff
  16. Steven S Rosenfeld
  17. Scott T Forth
  18. Jawdat Al-Bassam  Is a corresponding author
  1. University of California, Davis, United States
  2. The Scripps Research Institute, United States
  3. University of Vermont, United States
  4. Rensselaer Polytechnic Institute, United States
  5. Lerner Research Institute, Cleveland Clinic, United States
  6. Yale University, United States
  7. Mayo Clinic, United States
  8. Ben Gurion University of the Negev, Israel

Abstract

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains microtubule-bound states by slowing ATP-binding resulting in high-force production at both homotetramer ends.

Data availability

Two atomic coordinate files for Dm-KLP61F motor ATP-like MT(alpha-beta-tubulin) model is available at Protein Data Bank (PDB-ID: #XXXX. The Dm-KLP61F motor nucleotide-free MT(alpha-beta-tubulin) asymmetric unit Protein Data BankPDB-ID: #XXXXThe refined Dm-KLP61F motor AMPPNP MT cryo-EM map is available at the Electron microscopy Data bank (EMBD) EMDB ID:#XXXXand the Dm-KLP61F motor-tail nucleotide free MT cryo-EM map is available at Electron microscopy Data bank (EMBD) EMDB-iD:#XXXXDm-KLP61F motor-tail nucleotide free MT cryo-EM map (focused 3D-classification map) is available at the Electron microscopy Data bank (EMBD)EMDB-ID:#XXXX

Article and author information

Author details

  1. Tatyana Bodrug

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Elizabeth M Wilson-Kubalek

    Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Stanley Nithianantham

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6238-647X
  4. Alex F Thompson

    Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. April Alfieri

    Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ignas Gaska

    Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jennifer Major

    Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Garrett Debs

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Sayaka Inagaki

    Department of Pharmacology, Mayo Clinic, Jacksonville, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Pedro Gutierrez

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Larisa Gheber

    Department of Chemistry, Ben Gurion University of the Negev, Beer-Sheva, Israel
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3759-4001
  12. Richard J McKenney

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Charles Vaughn Sindelar

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6646-7776
  14. Ron Milligan

    Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Jason Stumpff

    Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Steven S Rosenfeld

    Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Scott T Forth

    Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, United States
    Competing interests
    The authors declare that no competing interests exist.
  18. Jawdat Al-Bassam

    Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
    For correspondence
    jmalbassam@ucdavis.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6625-2102

Funding

National Science Foundation (1615991)

  • Jawdat Al-Bassam

National Institutes of Health (GM110283)

  • Jawdat Al-Bassam

National Institutes of Health (GM121491)

  • Jason Stumpff

National Institutes of Health (GM130556)

  • Jason Stumpff

Israel Science Foundation (ISF 386/18)

  • Larisa Gheber

United States-Israel Binational Science Foundation (BSF-2015851)

  • Larisa Gheber

National Institutes of Health (GM130556)

  • Richard J McKenney

National Institutes of Health (GM052468)

  • Ron Milligan

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

Copyright

© 2020, Bodrug 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

  • 3,881
    views
  • 512
    downloads
  • 44
    citations

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

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. Tatyana Bodrug
  2. Elizabeth M Wilson-Kubalek
  3. Stanley Nithianantham
  4. Alex F Thompson
  5. April Alfieri
  6. Ignas Gaska
  7. Jennifer Major
  8. Garrett Debs
  9. Sayaka Inagaki
  10. Pedro Gutierrez
  11. Larisa Gheber
  12. Richard J McKenney
  13. Charles Vaughn Sindelar
  14. Ron Milligan
  15. Jason Stumpff
  16. Steven S Rosenfeld
  17. Scott T Forth
  18. Jawdat Al-Bassam
(2020)
The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding
eLife 9:e51131.
https://doi.org/10.7554/eLife.51131

Share this article

https://doi.org/10.7554/eLife.51131

Further reading

    1. Cancer Biology
    2. Cell Biology
    Ida Marie Boisen, Nadia Krarup Knudsen ... Martin Blomberg Jensen
    Research Article

    Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.

    1. Cell Biology
    2. Immunology and Inflammation
    Alejandro Rosell, Agata Adelajda Krygowska ... Esther Castellano Sanchez
    Research Article

    Macrophages are crucial in the body’s inflammatory response, with tightly regulated functions for optimal immune system performance. Our study reveals that the RAS–p110α signalling pathway, known for its involvement in various biological processes and tumourigenesis, regulates two vital aspects of the inflammatory response in macrophages: the initial monocyte movement and later-stage lysosomal function. Disrupting this pathway, either in a mouse model or through drug intervention, hampers the inflammatory response, leading to delayed resolution and the development of more severe acute inflammatory reactions in live models. This discovery uncovers a previously unknown role of the p110α isoform in immune regulation within macrophages, offering insight into the complex mechanisms governing their function during inflammation and opening new avenues for modulating inflammatory responses.