, Volume 148, Issue 2–3, pp 80–86 | Cite as

Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester

  • Seiji Sonobe
  • Hiroh Shibaoka


Actin filaments in cultured tobacco cells were stained by rhodamine-phalloidin after pretreatment with 100 μM m-maleidobenzoyl N-hydroxysuccinimide ester (MBS) followed by formaldehyde fixation. The use of MBS prior to formaldehyde fixation enabled us to visualize fine, transversely arranged cortical actin filaments in a majority of interphase tobacco cells. It also enabled us to double-stain fine actin filaments and microtubules in the same cells. The pattern of actin filaments and that of microtubules in the cortical region of a single tobacco cell bore a close resemblance to each other. The method which employed MBS was found to be useful also in visualizing fine cortical actin filaments in inner epidermal cells of onion bulbs.

Rhodamine-phalloidin seemed to induce the bundling of actin filaments both tobacco cells and in onion cells when it was applied to the cells which had not been subjected to fixation, indicating that the application of fluorescent-dye-labeled phallotoxins to unfixed cells involves the risk of observing artifically bundled actin filaments.


Protein cross-linking reagent Actin filaments Fluorescence microscopy m-Maleimidobenzoyl N-hydroxysuccinimide ester Protein fixation Tobacco BY-2 cell 



ethylenglycol-bis (β-aminoethyl ether)N,N,N′,N′-tetraacetic acid


dimethyl sulfoxide


m-maleimidobenzoyl N-hydroxysuccinimide ester


phosphate buffer saline


rhodamine-labeled phalloidin


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Brown RM Jr (1985) Cellulose microfibril assembly and orientation: recent development. J Cell Sci [Suppl] 2: 13–32Google Scholar
  2. Clayton L, Lloyd CW (1985) Actin organization during the cell cycle in meristematic plant cells. Exp Cell Res 156: 231–238PubMedGoogle Scholar
  3. Fukui Y, Katsumaru H (1980) Dynamics of nuclear actin bundle induction by dimethyl sulfoxide and factors affecting its development. J Cell Biol 84: 131–140PubMedGoogle Scholar
  4. Gorbsky GJ, Sammak PJ, Borisy GG (1988) Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol 106: 1185–1192PubMedGoogle Scholar
  5. Hawes CR (1985) Conventional and high voltage electron microscopy of the cytoskeleton and cytoplasmic matrix of carrot (Daucus carota L.) cells grown in suspension culture. Eur J Cell Biol 38: 201–210Google Scholar
  6. Kakimoto T, Shibaoka H (1987 a) Actin filaments and microtubules in the preprophase band and phragmoplast of tobacco cells. Protoplasma 140: 151–156Google Scholar
  7. — — (1987 b) A new method for preservation of actin filaments in higher plant cells. Plant Cell Physiol 28: 1581–1585Google Scholar
  8. Lancelle SA, Cresti M, Hepler KP (1987) Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes ofNicotiana alata. Protoplasma 140: 141–150Google Scholar
  9. Mitchison TJ, Kirschner MW (1985) Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol 101: 766–777PubMedGoogle Scholar
  10. Noguchi T, Ueda K (1988) Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida. Protoplasma 143: 188–192Google Scholar
  11. Palevitz BA (1987) Actin in the preprophase band ofAllium cepa. J Cell Biol 104: 1515–1519PubMedGoogle Scholar
  12. Parthasarathy MV, Perdue TD, Witztum A, Alvernaz J (1985) Actin network as a normal component of the cytoskeleton in many vascular plant cells. Am J Bot 72: 1318–1323Google Scholar
  13. Pesacreta TC, Carley WW, Webb WW, Parthasarathy MV (1982) F-actin in conifer roots. Proc Natl Acad Sci USA 79: 2898–2901Google Scholar
  14. Seagull RW, Falconer MM, Weerdenburg CA (1987) Microfilaments: dynamic arrays in higher plant cells. J Cell Biol 104: 995–1004PubMedGoogle Scholar
  15. —, Heath IB (1979) The effects of tannic acid on the in vivo preservation of microfilaments. Eur J Cell Biol 20: 184–188PubMedGoogle Scholar
  16. Sutoh K (1984) Actin-actin and actin-deoxyribonuclease I contact sites in the actin sequence. Biochemistry 23: 1942–1946PubMedGoogle Scholar
  17. Tiwari SC, Wick SM, Williamson RE, Gunning BES (1984) Cytoskeleton and integration of cellular function in cells of higher plants. J Cell Biol 99: 63s-69sPubMedGoogle Scholar
  18. Traas JA, Doonan JH, Rawlins DJ, Shaw PJ, Watts J, Lloyd CW (1987) An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus. J Cell Biol 105: 387–395PubMedGoogle Scholar
  19. Yahara I, Harada F, Sekita S, Yoshihira K, Natori S (1982) Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. J Cell Biol 92: 69–78PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Seiji Sonobe
    • 1
  • Hiroh Shibaoka
    • 1
  1. 1.Department of Biology, Faculty of ScienceOsaka UniversityJapan

Personalised recommendations