University of Massachusetts Medical School Faculty Publications
UMMS Affiliation
Program In Molecular Medicine
Publication Date
2017-07-12
Document Type
Article Preprint
Disciplines
Cell Biology | Cells | Fungi | Investigative Techniques
Abstract
Most available methods for nuclear isolation entail lengthy procedures that are difficult to master and generally emphasize yield and enrichment over nuclear preservation, thus limiting their utility for further studies. Here we demonstrate a novel and robust method to rapidly isolate well-preserved yeast nuclei. The method can be easily adapted to multiple preparation scales depending on experimental need and it can readily be performed on multiple samples by a single researcher in one day. We show that the nuclei fraction is strongly enriched and that the resulting nuclei are free from contaminating endoplasmic reticulum and other cell debris. EM studies show that preservation of nuclear morphology is exquisite, making it possible to study peripheral nuclear pore components such as the cytoplasmic filaments and the basket, whose structure is generally difficult to maintain ex vivo. In addition, incubation of isolated nuclei with bulk transport substrates of different sizes and with import cargo indicates that the nuclear envelope is intact and nuclear pores retain their capacity to bind transport substrates. Our results suggest that this preparation procedure will greatly facilitate studies of the yeast nucleus which have been difficult to establish and to multiplex to date.
Keywords
nuclear isolation, yeast nuclei, cytoplasmic filaments, basket, cell biology
Rights and Permissions
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
DOI of Published Version
10.1101/162388
Source
bioRxiv 162388; doi: https://doi.org/10.1101/162388. Link to preprint on bioRxiv service.
Journal/Book/Conference Title
bioRxiv
Repository Citation
Niepel M, Farr J, Rout MP, Strambio-De-Castilla C. (2017). Rapid isolation of functionally intact nuclei from the yeast Saccharomyces. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/162388. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1523
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.