Program In Molecular Medicine
Cell Biology | Cells | Fungi | Investigative Techniques
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.
nuclear isolation, yeast nuclei, cytoplasmic filaments, basket, cell biology
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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
bioRxiv 162388; doi: https://doi.org/10.1101/162388. Link to preprint on bioRxiv service.
Niepel M, Farr J, Rout MP, Strambio-De-Castillia C. (2017). Rapid isolation of functionally intact nuclei from the yeast Saccharomyces [preprint]. UMass Chan Medical School Faculty Publications. https://doi.org/10.1101/162388. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1523
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.