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. 1981 Feb 1;88(2):364–372. doi: 10.1083/jcb.88.2.364

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara

PMCID: PMC2111741  PMID: 6894146

Abstract

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.

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Selected References

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  1. Barak L. S., Yocum R. R., Nothnagel E. A., Webb W. W. Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. Proc Natl Acad Sci U S A. 1980 Feb;77(2):980–984. doi: 10.1073/pnas.77.2.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bottomley R. C., Trayer I. P. Affinity chromatography of immobilized actin and myosin. Biochem J. 1975 Aug;149(2):365–379. doi: 10.1042/bj1490365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradley M. O. Microfilaments and cytoplasmic streaming: inhibition of streaming with cytochalasin. J Cell Sci. 1973 Jan;12(1):327–343. doi: 10.1242/jcs.12.1.327. [DOI] [PubMed] [Google Scholar]
  4. Brenner S. L., Korn E. D. Substoichiometric concentrations of cytochalasin D inhibit actin polymerization. Additional evidence for an F-actin treadmill. J Biol Chem. 1979 Oct 25;254(20):9982–9985. [PubMed] [Google Scholar]
  5. Brown S. S., Spudich J. A. Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol. 1979 Dec;83(3):657–662. doi: 10.1083/jcb.83.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hartwig J. H., Stossel T. P. Cytochalasin B and the structure of actin gels. J Mol Biol. 1979 Nov 5;134(3):539–553. doi: 10.1016/0022-2836(79)90366-8. [DOI] [PubMed] [Google Scholar]
  7. Kamitsubo E. A 'window technique' for detailed observation of characean cytoplasmic streaming. Exp Cell Res. 1972 Oct;74(2):613–616. doi: 10.1016/0014-4827(72)90430-2. [DOI] [PubMed] [Google Scholar]
  8. Kato T., Tonomura Y. Identification of myosin in Nitella flexilis. J Biochem. 1977 Sep;82(3):777–782. doi: 10.1093/oxfordjournals.jbchem.a131754. [DOI] [PubMed] [Google Scholar]
  9. Kersey Y. M., Hepler P. K., Palevitz B. A., Wessells N. K. Polarity of actin filaments in Characean algae. Proc Natl Acad Sci U S A. 1976 Jan;73(1):165–167. doi: 10.1073/pnas.73.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kersey Y. M., Wessells N. K. Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study. J Cell Biol. 1976 Feb;68(2):264–275. doi: 10.1083/jcb.68.2.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Korn E. D. Biochemistry of actomyosin-dependent cell motility (a review). Proc Natl Acad Sci U S A. 1978 Feb;75(2):588–599. doi: 10.1073/pnas.75.2.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lin D. C., Tobin K. D., Grumet M., Lin S. Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation. J Cell Biol. 1980 Feb;84(2):455–460. doi: 10.1083/jcb.84.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Löw I., Dancker P., Wieland T. h. Stabilization of F-actin by phalloidin. Reversal of the destabilizing effect of cytochalasin B. FEBS Lett. 1975 Jun 15;54(2):263–265. doi: 10.1016/0014-5793(75)80088-3. [DOI] [PubMed] [Google Scholar]
  14. Nagai R., Hayama T. Ultrastructure of the endoplasmic factor responsible for cytoplasmic streaming in Chara internodal cells. J Cell Sci. 1979 Apr;36:121–136. doi: 10.1242/jcs.36.1.121. [DOI] [PubMed] [Google Scholar]
  15. Nagai R., Kamiya N. Differential treatment of Chara cells with cytochalasin B with special reference to its effect on cytoplasmic streaming. Exp Cell Res. 1977 Aug;108(1):231–237. [PubMed] [Google Scholar]
  16. Nagai R., Rebhun L. I. Cytoplasmic microfilaments in streaming Nitella cells. J Ultrastruct Res. 1966 Mar;14(5):571–589. doi: 10.1016/s0022-5320(66)80083-7. [DOI] [PubMed] [Google Scholar]
  17. Nothnagle E. A., Webb W. W. Barrier filter for fluorescence microscopy of strongly autofluorescent plant tissues. Application to actin cables in Chara. J Histochem Cytochem. 1979 May;27(5):1000–1002. doi: 10.1177/27.5.90068. [DOI] [PubMed] [Google Scholar]
  18. Palevitz B. A., Hepler P. K. Identification of actin in situ at the ectoplasm-endoplasm interface of Nitella. Microfilament-chloroplast association. J Cell Biol. 1975 Apr;65(1):29–38. doi: 10.1083/jcb.65.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanger J. W. Changing patterns of actin localization during cell division. Proc Natl Acad Sci U S A. 1975 May;72(5):1913–1916. doi: 10.1073/pnas.72.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stockem W., Weber K., Wehland J. The influence of microinjected phalloidin on locomotion, protoplasmic streaming and cytoplasmic organization in Amoeba proteus and Physarum polycephalum. Cytobiologie. 1978 Oct;18(1):114–131. [PubMed] [Google Scholar]
  21. Weber K., Rathke P. C., Osborn M., Franke W. W. Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB). Exp Cell Res. 1976 Oct 15;102(2):285–297. doi: 10.1016/0014-4827(76)90044-6. [DOI] [PubMed] [Google Scholar]
  22. Wehland J., Osborn M., Weber K. Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5613–5617. doi: 10.1073/pnas.74.12.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wehland J., Stockem W., Weber K. Cytoplasmic streaming in Amoeba proteus is inhibited by the actin-specific drug phalloidin. Exp Cell Res. 1978 Sep;115(2):451–454. doi: 10.1016/0014-4827(78)90307-5. [DOI] [PubMed] [Google Scholar]
  24. Wessells N. K., Spooner B. S., Ash J. F., Bradley M. O., Luduena M. A., Taylor E. L., Wrenn J. T., Yamada K. Microfilaments in cellular and developmental processes. Science. 1971 Jan 15;171(3967):135–143. doi: 10.1126/science.171.3967.135. [DOI] [PubMed] [Google Scholar]
  25. Wieland T., Govindan V. M. Phallotoxins bind to actins. FEBS Lett. 1974 Sep 15;46(1):351–353. doi: 10.1016/0014-5793(74)80404-7. [DOI] [PubMed] [Google Scholar]
  26. Williamson R. E. A light-microscope study of the action of cytochalasin B on the cells and isolated cytoplasm of the characeae. J Cell Sci. 1972 May;10(3):811–819. doi: 10.1242/jcs.10.3.811. [DOI] [PubMed] [Google Scholar]
  27. Williamson R. E. Cytochalasin B stabilises the sub-cortical actin bundles of Chara against a solution of low ionic strength. Cytobiologie. 1978 Oct;18(1):107–113. [PubMed] [Google Scholar]
  28. Williamson R. E. Cytoplasmic streaming in Chara: a cell model activated by ATP and inhibited by cytochalasin B. J Cell Sci. 1975 May;17(3):655–668. doi: 10.1242/jcs.17.3.655. [DOI] [PubMed] [Google Scholar]
  29. Williamson R. E. Filaments associated with the endoplasmic reticulum in the streaming cytoplasm of Chara corallina. Eur J Cell Biol. 1979 Dec;20(2):177–183. [PubMed] [Google Scholar]
  30. Yocum R. R. New laboratory scale purification of beta-amanitin from American Amanita phalloides. Biochemistry. 1978 Sep 5;17(18):3786–3789. doi: 10.1021/bi00611a018. [DOI] [PubMed] [Google Scholar]

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