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Serial Cryomicrotomy of Saccharomyces cerevisiae for Serial Electron Cryotomography

Ng, Cai Tong and Ladinsky, Mark S. and Gan, Lu (2020) Serial Cryomicrotomy of Saccharomyces cerevisiae for Serial Electron Cryotomography. Bio-protocol, 10 (22). Art. No. e3831. ISSN 2331-8325. https://resolver.caltech.edu/CaltechAUTHORS:20210309-075020738

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Abstract

Electron cryotomography (cryo-ET) is an increasingly popular technique to study cellular structures and macromolecules in situ. Due to poor penetration of electrons through thick biological samples, the vitreously frozen samples for cryo-ET need to be thin. For frozen-hydrated cells, such samples can be produced either by cryomicrotomy or cryo-FIB-milling. As a result, a tomogram of such a sample contains information of a small fraction of the entire cell volume, making it challenging to image rare structures in the cell or to determine the distribution of scattered structures. Here, we describe the tools and workflow that we designed to facilitate serial cryomicrotomy, which makes possible the exploration of a larger volume of individual cells at molecular resolution. We successfully used serial cryomicrotomy to locate and image the Dam1/DASH complex located at microtubule plus ends inside mitotic Saccharomyces cerevisiae cells.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.21769/bioprotoc.3831DOIArticle
ORCID:
AuthorORCID
Ng, Cai Tong0000-0002-1082-8667
Ladinsky, Mark S.0000-0002-1036-3513
Gan, Lu0000-0002-8685-4896
Additional Information:© 2020 The Authors; exclusive licensee Bio-protocol LLC. We thank Chen Chen for the initial cryomicrotomy set up in the lab, Gemma An for suggesting the use of parallel-bar EM grids, and Olivia Ma for her help in preparation of figures and feedback on the manuscript. We also thank members of the Gan lab for feedback and tips on cryomicrotomy. We thank Satoru Chomabayashi (Narishige) for help in modifying the MN-151 to increase the joystick travel and Eddie Lee (JEG Engineering Supplies) for fabricating the other custom tools. This protocol was used in Ng et al., 2019. C.T. Ng and L. Gan were funded by Singapore Ministry of Education grant MOE2018-T2-2-146. M.S. Ladinsky was funded by National Institutes of Health grant (2 P50 GM082545-08) to Pamela J. Bjorkman. The authors declare no competing financial interests.
Funders:
Funding AgencyGrant Number
Ministry of Education (Singapore)MOE2018-T2-2-146
NIH2 P50 GM082545-08
Subject Keywords:Serial electron cryotomography, Serial cryomicrotomy, Saccharomyces cerevisiae, Cryo-electron microscopy, Cell structural biology in situ
Issue or Number:22
Record Number:CaltechAUTHORS:20210309-075020738
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210309-075020738
Official Citation:Ng, C., Ladinsky, M. and Gan, L. (2020). Serial Cryomicrotomy of Saccharomyces cerevisiae for Serial Electron Cryotomography. Bio-protocol 10(22): e3831. DOI: 10.21769/BioProtoc.3831
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108357
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:10 Mar 2021 19:39
Last Modified:10 Mar 2021 19:39

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