Abstract
Mitotic spindle microtubules pull chromosomes toward each pole to generate two daughter cells. Proper spindle formation and function are required to prevent tumorigenesis and cell death. The fission yeast Schizosaccharomyces pombe has been widely used as a model organism to understand the molecular mechanism of mitosis due to its convenience in genetics, molecular biology, and cell biology. The development of fluorescent protein systems and microscopy enables us to investigate the “true” behavior of proteins in living fission yeast cells using a strain with a fluorescence-tagged gene under its native promoter. In this way the level of expression of tagged protein is similar to the level of wild-type nontagged protein. In this chapter we illustrate standard methods to generate strains expressing fluorescently tagged proteins and to observe them under the microscope. Specifically, we introduce a GFP-tubulin strain to analyze the dynamic behavior of spindle microtubules. Observation of GFP-tubulin under its native promoter has illuminated the process of kinetochore–microtubule attachment process in fission yeast.
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Acknowledgments
We thank Rafael E. Carazo-Salas for technical advice on microscopy and many invaluable discussions. We also thank Akira Yamashita for methods on meiosis, Miguel Angel Garcia and Kazuhide Asakawa for microscopy, Hiromi Maekawa and Elmar Schiebel for the 3GFP plasmid, Roger Tsien for providing mRFP and mCherry, and Kayoko Tanaka for transferring the mCherry plasmid. We are grateful to members of the Yeast Group on the third floor of the Lincoln’s Inn Fields Laboratories and to Masayuki Yamamoto for continuous support. M.S. was a recipient of JSPS postdoctoral fellowship for research abroad. The London Research Institute is supported by Cancer Research UK.
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Sato, M., Toya, M., Toda, T. (2009). Visualization of Fluorescence-Tagged Proteins in Fission Yeast: The Analysis of Mitotic Spindle Dynamics Using GFP-Tubulin Under the Native Promoter. In: McAinsh, A. (eds) Mitosis. Methods in Molecular Biology, vol 545. Humana Press. https://doi.org/10.1007/978-1-60327-993-2_11
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DOI: https://doi.org/10.1007/978-1-60327-993-2_11
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