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Procedures for Cultivation, Observation, and Conventional Experiments in Cyanidioschyzon merolae

  • Shinya MiyagishimaEmail author
  • Jong Lin Wei
Chapter

Abstract

Cyanidioschyzon merolae 10D was originally isolated from a mixture of hot spring water sampled in Naples, Italy. Currently, this strain is available in the Microbial Culture Collection at the National Institute of Environmental Studies in Japan. The strain has been cultured in 2× Allen’s medium or its derivatives. The optimal growth conditions for this strain are as follows: pH 2.5, 42 °C, and ~100 μmol photons m−2 s−1, allowing the cell density to reach ~5 × 108 cells mL−1. C. merolae can also grow slowly at >20 °C. We generally store stock cultures at room temperature or at 30 °C under a low light condition (~20 μmol photons m−2 s−1) or as frozen stock in liquid nitrogen. Cell cycle progression can be synchronized by subjecting the culture to a 12-h light/12-h dark cycle. In addition, cells can be arrested at the S or M phases by adding relevant inhibitors. The shapes of cells and chloroplasts are clearly observed by phase-contrast or differential interference contrast microscopy. Because C. merolae lacks a cell wall, cellular contents (e.g., DNA, RNA, and proteins) are easily extracted.

Keywords

Cell cycle Cell division Cyanidiales Cyanidioschyzon merolae Red algae Synchronous culture 

Notes

Acknowledgments

We thank Dr. Kuroiwa (Japan Women’s University), Drs. Tanaka and Imamura (Tokyo Institute of Technology), Dr. Misumi (Yamaguchi University), Dr. Yoshikawa (Tokyo University of Agriculture), and their lab members and members of Miyagishima lab in National Institute of Genetics for providing information on C. merolae experiments. Our study was partly supported by Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research 25251039 (to S.M.) and by the Core Research for Evolutional Science and Technology Program of the Japan Science and Technology Agency (to S.M.).

References

  1. Allen MB (1959) Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch Mikrobiol 32:270–277CrossRefPubMedGoogle Scholar
  2. Fujiwara T, Tanaka K et al (2013) Spatiotemporal dynamics of condensins I and II: evolutionary insights from the primitive red alga Cyanidioschyzon merolae. Mol Biol Cell 24:2515–2527CrossRefPubMedPubMedCentralGoogle Scholar
  3. Fujiwara T, Kanesaki Y et al (2015) A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae. Front Plant Sci 6:657PubMedPubMedCentralGoogle Scholar
  4. Imamura S, Kanesaki Y et al (2009) R2R3-type MYB transcription factor, CmMYB1, is a central nitrogen assimilation regulator in Cyanidioschyzon merolae. Proc Natl Acad Sci U S A 106:12548–12553CrossRefPubMedPubMedCentralGoogle Scholar
  5. Imamura S, Terashita M et al (2010) Nitrate assimilatory genes and their transcriptional regulation in a unicellular red alga Cyanidioschyzon merolae: genetic evidence for nitrite reduction by a sulfite reductase-like enzyme. Plant Cell Physiol 51:707–717CrossRefPubMedGoogle Scholar
  6. Imamura S, Ishiwata A et al (2013) Expression of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga Cyanidioschyzon merolae. Biochem Biophys Res Commun 439:264–269CrossRefPubMedGoogle Scholar
  7. Itoh R, Takahashi H et al (1996) Aphidicolin uncouples the chloroplast division cycle from the mitotic cycle in the unicellular red alga Cyanidioschyzon merolae. Eur J Cell Biol 71:303–310PubMedGoogle Scholar
  8. Matsuzaki M, Misumi O et al (2004) Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D. Nature 428:653–657CrossRefPubMedGoogle Scholar
  9. Minoda A, Sakagami R et al (2004) Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 45:667–671CrossRefPubMedGoogle Scholar
  10. Miyagishima SY, Itoh R et al (1999a) Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae. Planta 209:371–375CrossRefPubMedGoogle Scholar
  11. Miyagishima SY, Itoh R et al (1999b) Real-time analyses of chloroplast and mitochondrial division and differences in the behavior of their dividing rings during contraction. Planta 207:343–353CrossRefGoogle Scholar
  12. Miyagishima S, Takahara M et al (2001) Plastid division is driven by a complex mechanism that involves differential transition of the bacterial and eukaryotic division rings. Plant Cell 13:2257–2268CrossRefPubMedCentralGoogle Scholar
  13. Miyagishima SY, Nishida K et al (2003) A plant-specific dynamin-related protein forms a ring at the chloroplast division site. Plant Cell 15:655–665CrossRefPubMedPubMedCentralGoogle Scholar
  14. Miyagishima SY, Suzuki K et al (2012) Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle. Mol Biol Evol 29:2957–2970CrossRefPubMedGoogle Scholar
  15. Miyagishima SY, Fujiwara T et al (2014) Translation-independent circadian control of the cell cycle in a unicellular photosynthetic eukaryote. Nat Commun 5:3807CrossRefPubMedGoogle Scholar
  16. Nishida K, Yagisawa F et al (2005) Cell cycle-regulated, microtubule-independent organelle division in Cyanidioschyzon merolae. Mol Biol Cell 16:2493–2502CrossRefPubMedPubMedCentralGoogle Scholar
  17. Nishida K, Yagisawa F et al (2007) WD40 protein Mda1 is purified with Dnm1 and forms a dividing ring for mitochondria before Dnm1 in Cyanidioschyzon merolae. Proc Natl Acad Sci U S A 104:4736–4741CrossRefPubMedPubMedCentralGoogle Scholar
  18. Nozaki H, Takano H et al (2007) A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae. BMC Biol 5:28CrossRefPubMedPubMedCentralGoogle Scholar
  19. Ohnuma M, Yokoyama T et al (2008) Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant Cell Physiol 49:117–120CrossRefPubMedGoogle Scholar
  20. Ohnuma M, Kuroiwa T et al (2011) Optimization of cryopreservation conditions for the unicellular red alga Cyanidioschyzon merolae. J Gen Apple Microbiol 57:137–143CrossRefGoogle Scholar
  21. Sumiya N, Fujiwara T et al (2014) Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae. PLoS One 9:e111261CrossRefPubMedPubMedCentralGoogle Scholar
  22. Sumiya N, Fujiwara T et al (2016) Chloroplast division checkpoint in eukaryotic algae. Proc Natl Acad Sci U S A 113:E7629–E7638CrossRefPubMedPubMedCentralGoogle Scholar
  23. Suzuki K, Ehara T et al (1994) Behavior of mitochondria, chloroplasts and their nuclei during the mitotic cycle in the ultramicroalga Cyanidioschyzon merolae. Eur J Cell Biol 63:280–288PubMedGoogle Scholar
  24. Terui S, Suzuki K et al (1995) Synchronization of chloroplast division in the ultramicroalga Cyanidioschyzon merolae (Rhodophyta) by treatment with light and aphidicolin. J Phycol 31:958–961CrossRefGoogle Scholar
  25. Toda K, Takahashi H, Itoh R, Kuroiwa T (1995) DNA contents of cell nuclei in two Cyanidiophyceae: Cyanidioschyzon merolae and Cyanidium caldarium Forma A. Cytologia (Tokyo) 60:183–188CrossRefGoogle Scholar
  26. Yagisawa F, Nishida K et al (2009) Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae. Plant J 60:882–893CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Department of Cell GeneticsNational Institute of GeneticsMishimaJapan
  2. 2.Department of GeneticsGraduate University for Advanced StudiesShizuokaJapan

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