Immunocytochemical Analysis of α-Tubulin Distribution Before and After Rapid Axopodial Contraction in the Centrohelid Raphidocystis contractilis
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RIS BIB ENDNOTEImmunocytochemical Analysis of α-Tubulin Distribution Before and After Rapid Axopodial Contraction in the Centrohelid Raphidocystis contractilis
Publication date: 2020
Acta Protozoologica, 2020, Volume 59, Issue 1, pp. 1 - 12
https://doi.org/10.4467/16890027AP.20.001.12157Authors
Immunocytochemical Analysis of α-Tubulin Distribution Before and After Rapid Axopodial Contraction in the Centrohelid Raphidocystis contractilis
The centrohelid Raphidocystis contractilis is a heliozoan that has many radiating axopodia, each containing a bundle of microtubules. Although the rapid contraction of the axopodia at nearly a video rate (30 frames/s) is induced by mechanical stimuli, the mechanism underlying this phenomenon in R. contractilis has not yet been elucidated. In the present study, we described for the first time an adequate immunocytochemical fixation procedure for R. contractilis and the cellular distribution of α-tubulin before and after rapid axopodial contraction. We developed a flow-through chamber equipped with a micro-syringe pump that allowed the test solution to be injected at a flow rate below the threshold required to induce rapid axopodial contraction. Next, we used this injection method for evaluating the effects of different combinations of two fixatives (paraformaldehyde or glutaraldehyde) and two buffers (phosphate buffer or PHEM) on the morphological structure of the axopodia. A low concentration of glutaraldehyde in PHEM was identified as an adequate fixative for immunocytochemistry. The distribution of α-tubulin before and after rapid axopodial contraction was examined using immunocytochemistry and confocal laser scanning fluorescence microscopy. Positive signals were initially detected along the extended axopodia from the tips to the bases and were distributed in a non-uniform manner within the axopodia. Conversely, after the induction of a rapid axopodial contraction, these positive signals accumulated in the peripheral region of the cell. These results indicated that axopodial microtubules disassemble into fragments and/ or tubulin subunits during rapid axopodial contraction. Therefore, we hypothesize that the mechanism of extremely rapid axopodial contraction accompanied by cytoskeletal microtubule degradation in R. contractilis involves microtubule-severing at multiple sites.
Allan V. J. (1999) Basic immunofluorescence. In: Protein Localization by Fluorescence Microscopy: A Practical Approach, (Ed. V. J. Allan). Oxford University Press, Oxford, 1–26
Ando M., Shigenaka Y. (1989) Structure and function of the cytoskeleton in heliozoa: I. Mechanism of rapid axopodial contraction in Echinosphaerium. Cell Motil. Cytoskeleton 14: 288–301
Bardele C. F. (1975) The fine structure of the centrohelidian heliozoan Heterophrys marina. Cell Tissue Res. 161: 85–102
Belmont L. D., Hyman A. A., Sawin K. E., Mitchison T. J. (1990) Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62: 579–589
Cassimeris L., Pryer N. K., Salmon E. D. (1988) Real-time observations of microtubule dynamic instability in living cells. J. Cell Biol. 107: 2223–2231
Evangelio J. A., Abal M., Barasoain I., Souto A. A., Lillo M. P., Acuña A. U., Andreu J. M. (1998) Fluorescent taxoids as probes of the microtubule cytoskeleton. Cell Motil. Cytoskeleton 39: 73–90
Febvre-Chevalier C., Bilbaut A., Bone Q., Febvre J. (1986) Sodiumcalcium action potential associated with contraction in the heliozoan Actinocoryne contractilis. J. Exp. Biol. 122: 177–192
Febvre-Chevalier C., Febvre J. (1992) Microtubule dissassembly in vivo: intercalary destabilization and breakdown of microtubules in the heliozoan Actinocoryne contractilis. J. Cell Biol. 118: 585–594
Febvre-Chevalier C., Febvre J. (1993) Structural and physiological basis of axopodial dynamics. Acta Protozool. 32: 211–228
Febvre-Chevalier C., Febvre J. (2001) Heliozoa. In: eLS. John Wiley & Sons Ltd, Chichester, DOI: 10.1038/npg.els.0002103
Griffiths G. (2012) Preembedding Immuno-Labelling. In: Fine structure immunocytochemistry, (Ed. G. Griffiths). Springer, Heidelberg, 345–370
Gueth-Hallonet C., Antony C., Aghion J., Santa-Maria A., Lajoie-Mazenc I., Wright M., Maro B. (1993) Gamma-tubulin is present in acentriolar MTOCs during early mouse development. J. Cell Sci. 105: 157–166
Horio T., Hotani H. (1986) Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature 321: 605–607
Karabay A., Korulu Ş., Ünal A. Y. (2012) Immunocytochemistry of Cytoskeleton Proteins. In: Applications of Immunocytochemistry, (Ed. H. Dehghani). Intech Open Access Publisher, Rijeka, 97–1161
Khan S. M. K., Arikawa M., Omura G., Suetomo Y., Kakuta S., Suzaki, T. (2003) Axopodial contraction in the heliozoon Raphidiophrys contractilis requires extracellular Ca2+. Zool. Sci. 20: 1367–1373
Khan S. M. K., Yoshimura C., Arikawa M., Omura G., Nishiyama, S., Suetomo, Y., Suzaki, T. (2006) Axopodial degradation in the heliozoon Raphidiophrys contractilis: a novel bioassay system for detecting heavy metal toxicity in an aquatic environment. Environ. Sci. 13: 193–200
Kiernan J. A. (2000) Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Micros. Today 8: 8–13
Kinoshita E., Suzaki T., Shigenaka Y., Sugiyama M. (1995) Ultrastructure and rapid axopodial contraction of a heliozoa, Raphidiophrys contractilis sp. nov. J. Eukaryot. Microbiol. 42: 283–288
Kinoshita E., Shigenaka Y., Suzaki T. (2001) The ultrastructure of contractile tubules in the heliozoon Actinophrys sol and their possible involvement in rapid axopodial contraction. J. Eukaryot. Microbiol. 48: 519–526
Matsuoka T., Shigenaka Y., Naitoh Y. (1985) A model of contractile tubules showing how they contract in the heliozoan Echinosphaerium. Cell Struct. Funct. 10: 63–70
McBeath E., Fujiwara K. (1984) Improved fixation for immunofluorescence microscopy using light-activated 1, 3, 5-triazido-2, 4, 6-trinitrobenzene (TTB). J. Cell Biol. 99: 2061–2073
Moore R. C., Zhang M., Cassimeris L., Cyr R. J. (1997) In vitro assembled plant microtubules exhibit a high state of dynamic instability. Cell Motil. Cytoskeleton 38: 278–286
Ockleford C. D., Tucker J. B. (1973) Growth, breakdown, repair, and rapid contraction of microtubular axopodia in the heliozoan Actinophrys sol. J. Ultrastruct. Res. 44: 369–387
Peknicova J., Pexidrova M., Kubatova A., Koubek P., Tepla O., Sulimenko T., Draber P. (2007) Expression of beta-tubulin epitope in human sperm with pathological spermiogram. Fertil. Steril. 88: 1120–1128
Prigent Y., Kann M. L., Lach-Gar H., Pechart I., Fouquet J. P. (1996) Glutamylated tubulin as a marker of microtubule heterogeneity in the human sperm flagellum. Mol. Hum. Reprod. 2: 573–581
Sakaguchi M., Suzaki T., Khan S. M. K., Hausmann K. (2002) Food capture by kinetocysts in the heliozoon Raphidiophrys contractilis. Eur. J. Protistol. 37: 453–458
Schliwa M., Van Blerkom J. (1981) Structural interaction of cytoskeletal components. J. Cell Biol. 90: 222–235
Smith T. K., Lund E. K., Parker M. L., Clarke R. G., Johnson I. T. (2004) Allyl-isothiocyanate causes mitotic block, loss of cell adhesion and disrupted cytoskeletal structure in HT29 cells. Carcinogenesis 25: 1409–1415
Sudo H., Baas P. W. (2010) Acetylation of microtubules influences their sensitivity to severing by katanin in neurons and fibroblasts. J. Neurosci. 30: 7215–7226
Suzaki T., Ando M., Inai Y., Shigenaka Y. (1994) Structure and function of the cytoskeleton in heliozoa: 3. Rapid microtubule disorganization during axopodial contraction in Echinosphaerium. Eur. J. Protistol. 30: 404–413
Suzaki T., Shigenaka Y., Watanabe S., Toyohara A. (1980) Food capture and ingestion in the large heliozoan, Echinosphaerium nucleofilum. J. Cell Sci. 42: 61–79
Tilney L. G., Hiramoto Y., Marsland D. (1966) Studies on the microtubules in Heliozoa. III. A pressure analysis of the role of these structures in the formation and maintenance of the axopodia of Actinosphaerium nucleofilum (Barrett). J. Cell biol. 29: 77–95
Waters J. C., Chen R. H., Murray A. W., Salmon E. D. (1998) Localization of Mad2 to kinetochores depends on microtubule attachment, not tension. J. Cell Biol. 141: 1181–1191
Watters C. (1968) Studies on the Motility of the Heliozoa: I. The Locomotion of Actinosphaerium Eichhorni and Actinophrys sp. J. Cell Sci. 3: 231–244
Zlatogursky V. V., Drachko D., Klimov V. I., Shishkin, Y. (2018) On the phylogenetic position of the genus Raphidocystis (Haptista: Centroplasthelida) with notes on the dimorphism in centrohelid life cycle. Eur. J. Protistol. 64: 82–90
Received on 21st November, 2019; revised on 31st March, 2020; accepted on 23rd April, 2020
Information: Acta Protozoologica, 2020, Volume 59, Issue 1, pp. 1 - 12
Article type: Original article
Laboratory of Animal Physiology and Pharmacology, Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan
Laboratory of Cell Physiology, Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan
Laboratory of Cell Physiology, Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan
Laboratory of Cell Physiology, Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan
Laboratory of Animal Physiology and Pharmacology, Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan
Laboratory of Animal Physiology and Pharmacology, Department of Animal Science, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan
Laboratory of Cell Physiology, Department of Science Education, Graduate School of Education, Okayama University, Okayama 700-8530, Japan
Published at: 2020
Received at: 21.11.2019
Accepted at: 23.04.2020
Article status: Open
Licence: CC BY-NC-ND
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