Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ
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.
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.Attached Files
Published - 455.full.pdf
Supplemental Material - 49891_0_supp_598556_pfrw94.mov
Supplemental Material - 49891_0_supp_598557_pfrwx8.mp4.mp4
Files
| Name | Size | Download all |
|---|---|---|
|
md5:1b91334b480f06811d452d81952eca64
|
4.4 MB | Preview Download |
|
md5:e2e3babb09d0412f009b4fe52649622f
|
115.4 MB | Download |
|
md5:236578fb52746e58c7f72176bf76f1ee
|
227.6 MB | Download |
Additional details
- PMCID
- PMC6363454
- Eprint ID
- 92990
- Resolver ID
- CaltechAUTHORS:20190220-073843835
- R-154-000-515-133
- National University of Singapore
- R-154-000-524-651
- National University of Singapore
- D-E12-303-154-217
- National University of Singapore
- YIA R-154-000-558-133
- Ministry of Education (Singapore)
- Agency for Science, Technology and Research (A*STAR)
- Created
-
2019-02-20Created from EPrint's datestamp field
- Updated
-
2021-11-16Created from EPrint's last_modified field