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Synchronized fission yeast meiosis using an ATP analog–sensitive Pat1 protein kinase

Nature Protocols volume 9, pages 223–231 (2014)Cite this article

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Abstract

Synchronous cultures are often indispensable for studying meiosis. Here we present an optimized protocol for induction of synchronous meiosis in the fission yeast Schizosaccharomyces pombe. Chemical inactivation of an ATP analog–sensitive form of the Pat1 kinase (pat1-as2) by adding the ATP analog 1-NM-PP1 in G1-arrested cells allows the induction of synchronous meiosis at optimal temperature (25°C). Importantly, this protocol eliminates detrimental effects of elevated temperature (34°C), which is required to inactivate the commonly used temperature-sensitive Pat1 kinase mutant (pat1-114). The addition of the mat-Pc gene to a mat1-M strain further improves chromosome segregation and spore viability. Thus, our protocol offers highly synchronous meiosis at optimal temperature, with most characteristics similar to those of wild-type meiosis. The synchronization protocol can be completed in 5 d (not including strain production, which may take as long as 2 or 3 months).

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Figure 1: Zygotic and azygotic meiosis in the fission yeast S. pombe.
Figure 2: Synchronous meiosis induced by the inactivation of Pat1.
Figure 3: Sensitivity to ATP analogs of cells expressing Pat1-as2.

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Acknowledgements

This work was supported by Austrian Science Fund grants P23609 and P21437 and by the Slovak Research and Development Agency under contract nos. APVV-0111-12 and APVV-0334-12. Part of this research was supported by the US National Institutes of Health grant no. GM032194 to G.R.S. L.C. was supported by the (European Community's) Seventh Framework Programme (FP7/2007–2013) under grant agreement no. PERG07-GA-2010-268167. J.G. was supported by the (European Community's) Seventh Framework Programme (FP7/2007–2013) under grant agreement no. PCIG11-GA-2012-322300. S.P. was supported by A European Molecular Biology Organization (EMBO) long-term fellowship. We thank J. Ayte, A. Yamamoto, C. Zhang, H. Murakami, A. Lorenz and Z. Benko for helpful discussions.

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Author notes

  1. Lubos Cipak, Silvia Polakova and Randy W Hyppa: These authors contributed equally to this work.

Authors and Affiliations

  1. Max F. Perutz Laboratories, University of Vienna, Vienna, Austria

    Lubos Cipak, Silvia Polakova & Juraj Gregan

  2. Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia

    Lubos Cipak

  3. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Randy W Hyppa & Gerald R Smith

  4. Department of Genetics, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia

    Juraj Gregan

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  1. Lubos Cipak
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Contributions

J.G. and G.R.S. designed the experiments and wrote the manuscript. S.P., L.C. and R.W.H. performed the experiments and contributed to the writing of the manuscript. S.P., L.C. and R.W.H. contributed equally. Both J.G. and G.R.S. are corresponding authors.

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Correspondence to Gerald R Smith or Juraj Gregan.

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Cipak, L., Polakova, S., Hyppa, R. et al. Synchronized fission yeast meiosis using an ATP analog–sensitive Pat1 protein kinase. Nat Protoc 9, 223–231 (2014). https://doi.org/10.1038/nprot.2014.013

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