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

Author:

Sogame Yoichiro1,Kojima Katsuhiko2,Takeshita Toshikazu2,Kikuchi Shiho3,Shimada Yuto3,Nakamura Rikiya3,Arikawa Mikihiko3,Miyata Seiji4,Kinoshita Eiji5,Suizu Futoshi6,Matsuoka Tatsuomi3

Affiliation:

1. National Institute of Technology Fukushima College, Iwaki, Fukushima Japan

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

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

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

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

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

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.

Publisher

Uniwersytet Jagiellonski - Wydawnictwo Uniwersytetu Jagiellonskiego

Subject

General Agricultural and Biological Sciences

Reference34 articles.

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2. 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

3. 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

4. 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

5. Foissner W. (1993) Colpodea (Ciliophora). Gustav Fischer Verlag, Stuttgart

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