1. Cell Biology

Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

  1. Tae Yeon Yoo  Is a corresponding author
  2. Jeong-Mo Choi
  3. William Conway
  4. Che-Hang Yu
  5. Rohit V Pappu
  6. Daniel J Needleman
  1. Harvard University, United States
  2. Washington University in St Louis, United States
https://doi.org/10.7554/eLife.36392
Share this article
Cite this article
  1. Tae Yeon Yoo
  2. Jeong-Mo Choi
  3. William Conway
  4. Che-Hang Yu
  5. Rohit V Pappu
  6. Daniel J Needleman
(2018)
Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments
eLife 7:e36392.
https://doi.org/10.7554/eLife.36392
  2. Download BibTeX
  3. Download .RIS

Abstract

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells. We found that NDC80 binding is modulated in a chromosome autonomous fashion over prometaphase and metaphase, and is predominantly regulated by centromere tension. We show that this tension dependency requires phosphorylation of the N-terminal tail of Hec1, a component of the NDC80 complex, and the proper localization of Aurora B kinase, which modulates NDC80 binding. Our results lead to a mathematical model of the molecular basis of tension-dependent NDC80 binding to kinetochore microtubules in vivo.

Data availability

- All microscopy image data and data points in the presented plots have been deposited in Dryad (DOI: https://doi.org/10.5061/dryad.14rr125)- Analysis codes are deposited in Github, where doi's are provided in the manuscript.

The following data sets were generated

Article and author information

Author details

  1. Tae Yeon Yoo

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    For correspondence
    taeyeon_yoo@hms.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8145-1051

  2. Jeong-Mo Choi

    Department of Biomedical Engineering, Washington University in St Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2656-4851

  3. William Conway

    Department of Physics, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Che-Hang Yu

    John A Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0353-9752

  5. Rohit V Pappu

    Department of Biomedical Engineering, Washington University in St Louis, St Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2568-1378

  6. Daniel J Needleman

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Science Foundation (DBI-0959721)

  • Daniel J Needleman

National Institutes of Health (R01NS056114)

  • Rohit V Pappu

National Science Foundation (DMR-0820484)

  • Daniel J Needleman

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

Reviewing Editor

  1. Trisha N Davis, University of Washington, United States

Version history

  1. Received: March 5, 2018
  2. Accepted: July 24, 2018
  3. Accepted Manuscript published: July 25, 2018 (version 1)
  4. Version of Record published: August 13, 2018 (version 2)
  5. Version of Record updated: August 15, 2018 (version 3)
  6. Version of Record updated: August 23, 2018 (version 4)

Copyright

© 2018, Yoo 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

  • 3,129
    views
  • 571
    downloads
  • 47
    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. Tae Yeon Yoo
  2. Jeong-Mo Choi
  3. William Conway
  4. Che-Hang Yu
  5. Rohit V Pappu
  6. Daniel J Needleman
(2018)
Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments
eLife 7:e36392.
https://doi.org/10.7554/eLife.36392

Share this article

https://doi.org/10.7554/eLife.36392

Categories and tags

Research organism

Further reading

    1. Cell Biology

    A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α

    Joanne Tung, Lei Huang ... Adriana Ordonez
    Research Article

    Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

    1. Cancer Biology
    2. Cell Biology

    A syngeneic spontaneous zebrafish model of tp53-deficient, EGFRvIII, and PI3KCAH1047R-driven glioblastoma reveals inhibitory roles for inflammation during tumor initiation and relapse in vivo

    Alex Weiss, Cassandra D'Amata ... Madeline N Hayes
    Research Article

    High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFRvIII and PI3KCAH1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1:EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations.

    1. Cancer Biology
    2. Cell Biology

    Cancer: Modeling glioblastoma

    Ian Lorimer
    Insight

    Establishing a zebrafish model of a deadly type of brain tumor highlights the role of the immune system in the early stages of the disease.