1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

  1. Pim J Huis in 't Veld
  2. Vladimir A Volkov
  3. Isabelle D Stender
  4. Andrea Musacchio  Is a corresponding author
  5. Marileen Dogterom  Is a corresponding author
  1. Max Planck Institute of Molecular Physiology, Germany
  2. Delft University of Technology, Netherlands
https://doi.org/10.7554/eLife.49539
Share this article
Cite this article
  1. Pim J Huis in 't Veld
  2. Vladimir A Volkov
  3. Isabelle D Stender
  4. Andrea Musacchio
  5. Marileen Dogterom
(2019)
Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling
eLife 8:e49539.
https://doi.org/10.7554/eLife.49539
  2. Download BibTeX
  3. Download .RIS

Abstract

Errorless chromosome segregation requires load-bearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Both the Ndc80-tail, which we reveal to be essential for microtubule end-tracking, and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. Modulation of force-coupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.

Data availability

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

The following previously published data sets were used

Article and author information

Author details

  1. Pim J Huis in 't Veld

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0234-6390

  2. Vladimir A Volkov

    Department of Bionanoscience, Delft University of Technology, Delft, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5407-3366

  3. Isabelle D Stender

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    Competing interests
    The authors declare that no competing interests exist.

  4. Andrea Musacchio

    Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany
    For correspondence
    andrea.musacchio@mpi-dortmund.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2362-8784

  5. Marileen Dogterom

    Department of Bionanoscience, Delft University of Technology, Delft, Netherlands
    For correspondence
    m.dogterom@tudelft.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8803-5261

Funding

European Commission (ERC AdG RECEPIANCE (proposal 669686))

  • Andrea Musacchio

Deutsche Forschungsgemeinschaft (CRC1093)

  • Andrea Musacchio

European Commission (ERC SG MODELCELL (proposal 609822))

  • Marileen Dogterom

European Molecular Biology Organization (STF7203)

  • Pim J Huis in 't Veld

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

Copyright

© 2019, Huis in 't Veld 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

  • 2,505
    views
  • 414
    downloads
  • 42
    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. Pim J Huis in 't Veld
  2. Vladimir A Volkov
  3. Isabelle D Stender
  4. Andrea Musacchio
  5. Marileen Dogterom
(2019)
Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling
eLife 8:e49539.
https://doi.org/10.7554/eLife.49539

Share this article

https://doi.org/10.7554/eLife.49539

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

    Kristina Ehring, Sophia Friederike Ehlers ... Kay Grobe
    Research Article

    The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    Point of View: Teaching troubleshooting skills to graduate students

    Gina Partipilo, Yang Gao ... Benjamin K Keitz
    Feature Article

    Troubleshooting is an important part of experimental research, but graduate students rarely receive formal training in this skill. In this article, we describe an initiative called Pipettes and Problem Solving that we developed to teach troubleshooting skills to graduate students at the University of Texas at Austin. An experienced researcher presents details of a hypothetical experiment that has produced unexpected results, and students have to propose new experiments that will help identify the source of the problem. We also provide slides and other resources that can be used to facilitate problem solving and teach troubleshooting skills at other institutions.

    1. Biochemistry and Chemical Biology
    2. Plant Biology

    Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production

    Hao Wang, Biying Zhu ... Zhaoliang Zhang
    Research Article

    Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.