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.

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

  • 3,247
    views
  • 581
    downloads
  • 52
    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

    Small-molecule activation of TFEB alleviates Niemann–Pick disease type C via promoting lysosomal exocytosis and biogenesis

    Kaili Du, Hongyu Chen ... Dan Li
    Research Article

    Niemann–Pick disease type C (NPC) is a devastating lysosomal storage disease characterized by abnormal cholesterol accumulation in lysosomes. Currently, there is no treatment for NPC. Transcription factor EB (TFEB), a member of the microphthalmia transcription factors (MiTF), has emerged as a master regulator of lysosomal function and promoted the clearance of substrates stored in cells. However, it is not known whether TFEB plays a role in cholesterol clearance in NPC disease. Here, we show that transgenic overexpression of TFEB, but not TFE3 (another member of MiTF family) facilitates cholesterol clearance in various NPC1 cell models. Pharmacological activation of TFEB by sulforaphane (SFN), a previously identified natural small-molecule TFEB agonist by us, can dramatically ameliorate cholesterol accumulation in human and mouse NPC1 cell models. In NPC1 cells, SFN induces TFEB nuclear translocation via a ROS-Ca2+-calcineurin-dependent but MTOR-independent pathway and upregulates the expression of TFEB-downstream genes, promoting lysosomal exocytosis and biogenesis. While genetic inhibition of TFEB abolishes the cholesterol clearance and exocytosis effect by SFN. In the NPC1 mouse model, SFN dephosphorylates/activates TFEB in the brain and exhibits potent efficacy of rescuing the loss of Purkinje cells and body weight. Hence, pharmacological upregulating lysosome machinery via targeting TFEB represents a promising approach to treat NPC and related lysosomal storage diseases, and provides the possibility of TFEB agonists, that is, SFN as potential NPC therapeutic candidates.

    1. Cell Biology

    Stratification of enterochromaffin cells by single-cell expression analysis

    Yan Song, Linda J Fothergill ... Gene W Yeo
    Research Article

    Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify 14 EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic, and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.

    1. Cell Biology
    2. Developmental Biology

    Lens Development: Bringing signaling complexity into focus

    Sarah Y Coomson, Salil A Lachke
    Insight

    A study in mice reveals key interactions between proteins involved in fibroblast growth factor signaling and how they contribute to distinct stages of eye lens development.