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

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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

1. Yoo TY
2. Choi J
3. Conway W
4. Yu C
5. Pappu RV
6. Needleman DJ
(2018) Data from: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments.
Available at Dryad Digital Repository under a CC0 Public Domain Dedication.

https://dx.doi.org/10.5061/dryad.14rr125

Article and author information

Author details

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.

"This ORCID iD identifies the author of this article:" 0000-0002-8145-1051
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.

"This ORCID iD identifies the author of this article:" 0000-0003-2656-4851
William Conway

Department of Physics, Harvard University, Cambridge, United States

Competing interests
The authors declare that no competing interests exist.
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.

"This ORCID iD identifies the author of this article:" 0000-0002-0353-9752
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.

"This ORCID iD identifies the author of this article:" 0000-0003-2568-1378
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

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.