Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity

  1. Breane G Budaitis
  2. Shashank Jariwala
  3. Dana N Reinemann
  4. Kristin I Schimert
  5. Guido Scarabelli
  6. Barry J Grant
  7. David Sept
  8. Matthew J Lang
  9. Kristen J Verhey  Is a corresponding author
  1. University of Michigan, United States
  2. Vanderbilt University, United States
  3. University of California, San Diego, United States

Abstract

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Breane G Budaitis

    Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Shashank Jariwala

    Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Dana N Reinemann

    Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Kristin I Schimert

    Biophysics Program, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9209-7986
  5. Guido Scarabelli

    Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Barry J Grant

    Division of Biological Sciences, Section of Molecular Biology, University of California, San Diego, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. David Sept

    Department of Biomedical Engineering, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Matthew J Lang

    Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Kristen J Verhey

    Cell and Developmental Biology Program, University of Michigan, Ann Arbor, United States
    For correspondence
    kjverhey@umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9329-4981

Funding

National Institutes of Health (R01GM070862)

  • Barry J Grant

National Science Foundation (1330792)

  • Matthew J Lang

Qatar Leadership Program (R35 GM130293)

  • Shashank Jariwala

National Science Foundation (1256260)

  • Breane G Budaitis

National Science Foundation (1445197)

  • Dana N Reinemann

National Institutes of Health (T32GM007315)

  • Breane G Budaitis

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

Reviewing Editor

  1. Thomas Surrey, The Francis Crick Institute, United Kingdom

Version history

  1. Received: December 5, 2018
  2. Accepted: May 9, 2019
  3. Accepted Manuscript published: May 14, 2019 (version 1)
  4. Version of Record published: May 23, 2019 (version 2)

Copyright

© 2019, Budaitis 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

  • 2,944
    views
  • 372
    downloads
  • 32
    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. Breane G Budaitis
  2. Shashank Jariwala
  3. Dana N Reinemann
  4. Kristin I Schimert
  5. Guido Scarabelli
  6. Barry J Grant
  7. David Sept
  8. Matthew J Lang
  9. Kristen J Verhey
(2019)
Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity
eLife 8:e44146.
https://doi.org/10.7554/eLife.44146

Share this article

https://doi.org/10.7554/eLife.44146

Further reading

    1. Cell Biology
    Gang Liu, Yunxuan Hou ... Xiumei Jiang
    Research Article

    Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron–sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron–sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Christopher TA Lewis, Elise G Melhedegaard ... Julien Ochala
    Research Article

    Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77–107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.