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A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

Nature Protocols volume 2, pages 1943–1953 (2007)Cite this article

Abstract

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.

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Figure 1: Assembly of microtube chambers.
Figure 2: Visualization of yeast cells, spheroplasts and isolated nuclei by light microscopy.
Figure 3: Diagram of the yeast nuclear isolation procedure by spheroplast centrifugation onto a scanning electron microscopy (SEM) silicon chip.
Figure 4: A schematic model demonstrating the freeze-grinding technique for nuclear isolation from yeast cells.
Figure 5: Morphology of whole cells, spheroplasts and nuclei of Saccharomyces cerevisiae visualized by scanning electron microscopy.
Figure 6: Nuclear pore complexes (NPCs) in the nuclear envelope (NE) of Saccharomyces cerevisiae visualized by scanning electron microscopy.
Figure 7: Morphology of the spheroplasts and nuclei of Saccharomyces pombe visualized by scanning electron microscopy.
Figure 8: Visualization of free nuclei isolated from Saccharomyces cerevisiae spheroplasts by homogenization.
Figure 9: Immuno-scanning electron microscopy (immuno-SEM) visualization of the anti-Nup116 Abs on the cytoplasmic surface of a Saccharomyces cerevisiae nucleus.
Figure 10: Visualization of nuclei isolated from frozen and ground budding yeast cells.

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Acknowledgements

T.D.A, S.A.R., S.M. and S.P.D. would like to acknowledge the support of Cancer Research (CR)-UK. E.K. acknowledges The Wellcome Trust and Russian Federation for Basic Research, M.W.G acknowledges The Wellcome Trust, and F.G. acknowledges the MRC. The authors thank Professor M. Rout (The Rockefeller University, New York, NY) for his advice in isolation of nuclei from frozen yeast.

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Authors and Affiliations

  1. Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090, Russia

    Elena Kiseleva & Ksenia Morozova

  2. Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, M20 4BX, Manchester, UK

    Terry D Allen, Sandra A Rutherford, Steve Murray, Fiona Gardiner & Sheona P Drummond

  3. School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK

    Martin W Goldberg

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  1. Elena Kiseleva
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Correspondence to Elena Kiseleva.

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Kiseleva, E., Allen, T., Rutherford, S. et al. A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM). Nat Protoc 2, 1943–1953 (2007). https://doi.org/10.1038/nprot.2007.251

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