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Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

Ng, Cai Tong and Deng, Li and Chen, Chen and Lim, Hong Hwa and Shi, Jian and Surana, Uttam and Gan, Lu (2019) Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ. Journal of Cell Biology, 218 (2). pp. 455-473. ISSN 0021-9525. PMCID PMC6363454. http://resolver.caltech.edu/CaltechAUTHORS:20190220-073843835

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Abstract

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1083/jcb.201809088DOIArticle
http://jcb.rupress.org/content/218/2/455PublisherArticle
http://jcb.rupress.org/content/suppl/2018/10/19/jcb.201809088.DC1PublisherSupplementary Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363454/PubMed CentralArticle
ORCID:
AuthorORCID
Ng, Cai Tong0000-0002-1082-8667
Lim, Hong Hwa0000-0002-0951-7220
Shi, Jian0000-0003-3810-5302
Surana, Uttam0000-0002-4785-6514
Gan, Lu0000-0002-8685-4896
Additional Information:© 2018 Ng et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http:// www .rupress .org/ terms/ ). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https:// creativecommons .org/ licenses/ by -nc -sa/ 4 .0/). Submitted: 26 September 2018; Revised: 25 October 2018; Accepted: 31 October 2018. We thank the CBIS microscopy staff for support and training, Gemma An for suggesting the use of parallel-bar grids, Shujun Cai for helping with cryo-EM, Simon Jenni and Steve Harrison for Dam1C/DASH plasmids and sharing results before publication, Jeff Yong for advice on chromatography, the Jensen laboratory for computer access, and members of the Gan group, Jack Johnson, Steve Harrison, Paul Matsudaira, and Kerry Bloom for feedback. C.T. Ng, C. Chen, L. Deng, and L. Gan were funded by National University of Singapore (NUS) startups R-154-000-515-133, R-154-000-524-651, and D-E12-303-154-217, a Ministry of Education grant T2 R-154-000-624-112, with equipment support from NUS (grant YIA R-154-000-558-133). HHL and US were funded by the Biomedical Research Council of A*STAR (Agency of Science Technology and Research), Singapore. The authors declare no competing financial interests. Author contributions: C.T. Ng, experiments, project design, and writing; L. Deng, experiments; C. Chen, project design and experiments; H.H. Lim, experiments; J. Shi, training; U. Surana, project design and writing; L. Gan, experiments, project design, and writing.
Funders:
Funding AgencyGrant Number
National University of SingaporeR-154-000-515-133
National University of SingaporeR-154-000-524-651
National University of SingaporeD-E12-303-154-217
Ministry of Education (Singapore)YIA R-154-000-558-133
Agency for Science, Technology and Research (A*STAR)UNSPECIFIED
PubMed Central ID:PMC6363454
Record Number:CaltechAUTHORS:20190220-073843835
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20190220-073843835
Official Citation:Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ. Cai Tong Ng, Li Deng, Chen Chen, Hong Hwa Lim, Jian Shi, Uttam Surana, Lu Gan. J Cell Biol Feb 2019, 218 (2) 455-473; DOI: 10.1083/jcb.201809088.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92990
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:20 Feb 2019 16:03
Last Modified:20 Feb 2019 18:45

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