Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

  1. David M Kern
  2. Julie K Monda
  3. Kuan-Chung Su
  4. Elizabeth M Wilson-Kubalek
  5. Iain M Cheeseman  Is a corresponding author
  1. Whitehead Institute, United States
  2. The Scripps Research Institute, United States

Abstract

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

Article and author information

Author details

  1. David M Kern

    Department of Biology, MIT, Whitehead Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8529-9045
  2. Julie K Monda

    Department of Biology, MIT, Whitehead Institute, Cambridge, MA, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Kuan-Chung Su

    Department of Biology, MIT, Whitehead Institute, Cambridge, MA, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Elizabeth M Wilson-Kubalek

    Department of Cell Biology, The Scripps Research Institute, La Jolla, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Iain M Cheeseman

    Department of Biology, MIT, Whitehead Institute, Cambridge, MA, United States
    For correspondence
    icheese@wi.mit.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3829-5612

Funding

National Institute of General Medical Sciences (GM088313)

  • Iain M Cheeseman

Leukemia and Lymphoma Society (Scholar Award)

  • Iain M Cheeseman

National Institute of General Medical Sciences (GM052468)

  • Elizabeth M Wilson-Kubalek

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

Reviewing Editor

  1. Jon Pines, The Gurdon Institute, United Kingdom

Version history

  1. Received: March 16, 2017
  2. Accepted: August 24, 2017
  3. Accepted Manuscript published: August 25, 2017 (version 1)
  4. Version of Record published: September 18, 2017 (version 2)
  5. Version of Record updated: September 21, 2017 (version 3)

Copyright

© 2017, Kern 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

  • 3,274
    Page views
  • 490
    Downloads
  • 28
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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. David M Kern
  2. Julie K Monda
  3. Kuan-Chung Su
  4. Elizabeth M Wilson-Kubalek
  5. Iain M Cheeseman
(2017)
Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80
eLife 6:e26866.
https://doi.org/10.7554/eLife.26866

Share this article

https://doi.org/10.7554/eLife.26866

Further reading

    1. Biochemistry and Chemical Biology
    2. Plant Biology
    Pradeep Kumar, Ankit Roy ... Rajan Sankaranarayanan
    Research Article

    Aldehydes, being an integral part of carbon metabolism, energy generation, and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes, i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Greg J Dodge, Alyssa J Anderson ... Barbara Imperiali
    Research Article

    Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.