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Analysis of Water-Soluble Proteins by Two-Dimensional Electrophoresis in the Encystment Process of Colpoda cucullus Nag-1 and Cytoskeletal Dynamics

Publication date: 17.03.2021

Acta Protozoologica, 2020, Volume 59, Issue 3-4, pp. 107 - 120

https://doi.org/10.4467/16890027AP.20.009.13264

Authors

,
Yoichiro Sogame
National Institute of Technology Fukushima College, Iwaki, Fukushima Japan
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,
Katsuhiko Kojima
Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
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,
Toshikazu Takeshita
Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
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,
Shiho Kikuchi
Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan
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,
Yuto Shimada
Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan
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,
Rikiya Nakamura
Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan
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,
Mikihiko Arikawa
Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan
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,
Seiji Miyata
Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan
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,
Eiji Kinoshita
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan
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,
Futoshi Suizu
Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
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Tatsuomi Matsuoka
Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan
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Abstract

Assays of protein contained in water-soluble fraction of encysting cells Colpoda cucullus Nag-1 by two-dimensional electrophoresis (2-D PAGE) and mass spectrometry (MS) revealed that the amount of β-tubulin abruptly increased in 2.5–10 h after encystment induction. Judging from the results that total α-tubulin content did not decrease much until 12 h after encystment induction, the result indicates that disassembly of microtubules may occur soon after encystment is induced. Therefore, we tried to visualize dynamics of microtubules. Immunofluorescence microscopy using anti-α-tubulin antibody indicated that disassembly of axonemal microtubules of cilia became within 1.5 h after encystment induction, and resorbed in 3 days. Although the cytoplasmic microtubules failed to be visualized clearly, encystmentdependent globulation of cells was promoted by taxol, an inhibitor of disassembly of microtubules. It is possible that a temporary formation of cytoplasmic microtubules may be involved in cell globulation.

The phosphorylation level of actin (43 kDa) became slightly elevated just after encystment induction. Lepidosomes, the sticky small globes surrounding encysting cells, were vividly stained with Acti-stain 555 phalloidin, suggesting that 43-kDa actin or its homologues may be contained in lepidosomes.

References

Asami H., Ohtani Y., Iino R., Sogame Y., Matsuoka T. (2010) Behavior and Ca2+-induced cell signaling for encystment of Colpoda cucullus. J. Protozool. Res. 20: 1−6
Chen J., Gao X., Wang B., Chen F., Wu N., Zhang Y. (2014) Proteomic approach to reveal the proteins associated with encystment of the ciliate Euplotes encysticus. PLOS ONE e97362
Constantin B., Meerschaert K., Vandekerckhove J., Gettemans J. (1998) Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions. J. Cell Sci. 111: 1695−1706
Defeu Soufo H. J., Reimold C., Linne U., Knust T., Gescher J., Graumann P. L. (2010) Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proc. Natl. Acad. Sci. (USA) 107: 3163−3168
Foissner W. (1993) Colpodea (Ciliophora). Gustav Fischer Verlag, Stuttgart
Foissner W., Stoeck T, Agatha S., Dunthorn M. (2011) Intraclass evolution and classification of the Colpodea (Ciliophora). J. Eukaryot. Microbiol. 58: 397−415
Funadani R., Sogame Y., Kojima K., Takeshita T., Yamamoto K., Tsujizono T., Suizu F., Miyata S., Yagyu K., Suzuki T., Arikawa M., Matsuoka T. (2016) Morphogenetic and molecular analyses of cyst wall components in the ciliated protozoan Colpoda cucullus Nag-1. FEMS Microbiol. Lett. 363: fnw203
Funatani R., Kida A., Watoh T., Matsuoka T. (2010) Morphological events during resting cyst formation (encystment) in the ciliated protozoan Colpoda cucullus. Protistology 6: 204−217.
Furuhasi K., Hatano S. (1990) Control of actin filament length by phosphorylation of fragmin-actin complex. J. Cell Biol. 111: 1081−1087
Jiang C., Wei W., Yan G., Shi T., Miao W. (2019) Transcriptome analysis reveals the molecular mechanism of resting cyst formation in Colpoda aspera. J. Eukaryot. Microbiol. 66: 212−220
Jones J. F., Carballido-López R., Errington J. (2001) Control of cell shape in bacteria: Helical, actin-like filaments in Bacillus subtilis. Cell 104: 913−922
Herrera-Martínez M., Hernández-Ramírez V. I., Lagunes-Guillén A. E., Chávez-Munguía B., Talamás-Rohana P. (2013) Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens. BioMed Res. Int. 2013: 919345
Kinoshita E., Kinoshita-Kikuta E., Takiyama K., Koike T. (2006) Phosphate-binding tag: A new tool to visualized phosphorylated proteins. Mol. Cell. Proteomics 5: 749−757
Laemmli U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680−685
MacLean-Fletcher S., Pollard T. D. (1980) Mechanism of action of cytochalasin B on actin. Cell 20: 329−341
Maeda H., Akematsu T., Fukui R., Matsuoka T. (2005) Studies on the resting cyst of ciliated protozoan Colpoda cucullus: Resistance to temperature and additional inducing factors for en-or excystment. J. Protozool. Res. 15: 7−13
Matsuoka K., Funadani R., Matsuoka T. (2017) Tolerance of Colpoda cucullus resting cysts to ultraviolet irradiation. J. Protozool. Res. 27: 1−7
Matsuoka T., Kondoh A., Sabashi K., Nagano N., Akematsu T., Kida A., Iino R. (2009) Role of Ca2+ and cAMP in a cell signaling pathway for resting cyst formation of ciliated protozoan Colpoda cucullus. Protistology 6: 103−110
Mirvis M., Stearns T., Nelson W. J. (2018) Cilium structure, assembly, and disassembly regulated by the cytoskeleton. Biochem. J. 475: 2329−2353
Müller H., Achilles-Day U. E. M., Day J. G. (2010) Tolerance of the resting cysts of Colpoda inflata (Ciliophora, Colpodea) and Meseres corlissi (Ciliophora, Spirotrichea) to desiccation and freezing. Eur. J. Protistol. 46: 133−142
Ohta Y., Akiyama T., Nishida E., Sakai H. (1987) Protein kinase C and cAMP-dependent protein kinase induce opposite effects on actin polymerizability. FEBS Lett. 222: 305−310
Pan N., Niu T., Bhatti M. Z., Zhang H., Fan X., Ni B., Chen J. (2019) Novel insights into molecular mechanisms of Pseudourostyla cristata encystment using comparative transcriptomics. Sci. Rep. 9: 19109
Phua S. C., Chiba S., Suzuki M., Su E., Roberson E. C., Pusapati G. V., Setou M., Rohatgi R., Reiter J. F., Ikegami K., Inoue T. (2017) Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell 168: 264−279, e15
Risinger A. L., Riffle S. M., Lopus M., Jordan M. A., Wilson L., Mooberry S. L. (2014) The taccalonolides and paclitaxel cause distinct effects on microtubule dynamics and aster formation. Mol. Cancer 28: 13−41
Sackett D. L., Wolff J. (1986) Proteolysis of tubulin and the substructure of the tubulin dimer. J. Biol. Chem. 261: 9070−9076
Schiff P. B., Fant J., Horwitz S. B. (1979) Promotion of microtubule assembly in vitro by taxol. Nature 277: 665−667
Sogame Y., Kida A., Matsuoka T. (2011a) Possible involvement of endocyst in tolerance of the resting cyst of Colpoda cucullus against HCl. African J. Microbiol. Res. 5: 4316−4320
Sogame Y., Kinoshita E., Matsuoka T. (2011b) Ca2+-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus. Eur. J. Protistol. 47: 208−213
Sogame Y., Kojima K., Takeshita T., Kinoshita E., Matsuoka T. (2012) EF-1αand mitochondrial ATP synthase βchain: Alteration of their expression in encystment-induced Colpoda cucullus. J. Euk. Microbiol. 59: 401−406
Sogame Y., Kojima K., Takeshita T., Kinoshita E., Matsuoka T. (2014a) Identification of cAMP-dependent phosphorylated proteins involved in the formation of environment-resistant resting cysts by the terrestrial ciliate Colpoda cucullus. Inv. Surv. J. 11: 213−218
Sogame Y., Kojima K., Takeshita T., Kinoshita E., Matsuoka T. (2014b) Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis. J. Eukariot. Microbiol. 61: 51−60
Sogame Y., Matsuoka T. (2013) Evaluation of intracellular Ca2+ concentration by fura 2 ratiometry in encystment-induced Colpoda cucullus. Acta Protozool. 52: 51−54
Taylor C. V., Strickland A. G. R. (1936) Effects of high vacua and extreme temperatures on the cysts of Colpoda cucullus. Physiol. Zool. 9: 15−26
Watoh T., Sekida S., Yamamoto K., Kida A., Matsuoka T. (2005) Morphological study on the encystment of the ciliated protozoan Colpoda cucullus. J. Protozool. Res. 15: 20−28

Information

Information: Acta Protozoologica, 2020, pp. 107 - 120

Article type: Original research article

Authors

National Institute of Technology Fukushima College, Iwaki, Fukushima Japan

Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan

Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan

Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan

Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan

Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan

Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan

Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan

Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan

Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

Department of Biological Science, Faculty of Science, Kochi University, Kochi, Japan

Published at: 17.03.2021

Received at: 22.04.2020

Accepted at: 05.08.2020

Article status: Open

Licence: CC BY-NC-ND  licence icon

Percentage share of authors:

Yoichiro Sogame (Author) - 9%
Katsuhiko Kojima (Author) - 9%
Toshikazu Takeshita (Author) - 9%
Shiho Kikuchi (Author) - 9%
Yuto Shimada (Author) - 9%
Rikiya Nakamura (Author) - 9%
Mikihiko Arikawa (Author) - 9%
Seiji Miyata (Author) - 9%
Eiji Kinoshita (Author) - 9%
Futoshi Suizu (Author) - 9%
Tatsuomi Matsuoka (Author) - 10%

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