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Rhodamine-phalloidin and anti-tubulin antibody staining of spindle fibres that were irradiated with an ultraviolet microbeam

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Summary

We irradiated chromosomal spindle fibres in crane-fly spermatocytes with an ultraviolet microbeam of 270 nm wavelength light with total energies near those that cause actin filaments in myofibrils to depolymerize; after irradiation we stained the cells with rhodamine-labelled phalloidin and with anti-tubulin antibodies. In some cells, the irradiation reduced both phalloidin and tubulin staining of the chromosomal spindle fibres; in other cells, the irradiations reduced phalloidin staining but not tubulin staining; in yet other cells, the irradiations reduced tubulin staining but not phalloidin staining. In all irradiated cells in which phalloidin staining was reduced in the irradiated areas phalloidin staining also was reduced poleward from the irradiated areas. These results show that phalloidin staining of chromosomal spindle fibres is not dependent on the presence of kinetochore microtubules, and, therefore, that actin filaments are present in the spindle fibres in vivo. We suggest that actin filaments present in spindle fibres in vivo may be involved in causing chromosome movements during anaphase.

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References

  • Aronson JF (1965) The use of fluorescein-labelled heavy meromyosin for the cytological demonstration of actin. J Cell Biol 26: 293–298

    PubMed  Google Scholar 

  • Barak LS, Nothnagel EA, Demarco EF, Webb WW (1981) Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement? Proc Natl Acad Sci USA 78: 3034–3038

    PubMed  Google Scholar 

  • Bajer AS (1973) Interaction of microtubules and the mechanism of chromosome movement (zipper hypothesis). 1. General principle. Cytobios 8: 139–160

    PubMed  Google Scholar 

  • Callaini G, Dallai R, Riparbelli MG (1993) F-actin domains in the syncytial blastoderm of the dipteranCeratitis capitala. J Cell Sci 104: 97–104

    Google Scholar 

  • Cassimeris LU, Walker RA, Pryer NK, Salmon ED (1987) Dynamic instability of microtubules. BioEssays 7: 149–154

    PubMed  Google Scholar 

  • Cline CA, Schatten G (1986) Microfilaments during sea urchin fertilization: fluorescence detection with rhodaminyl phalloidin. Gamete Res 14: 277–291

    PubMed  Google Scholar 

  • Czaban BB, Forer A (1985) The kinetic polarities of spindle microtubules in vivo, in crane fly spermatocytes. I. Kinetochore microtubules that re-form after treatment with colcemid. J Cell Sci 79: 1–37

    PubMed  Google Scholar 

  • — — (1991) Visualization of ultraviolet microbeam irradiation sites with uranyl acetate. J Microsc 164: 61–65

    Google Scholar 

  • — — (1992) Rhodamine-labelled phalloidin stains components in the chromosomal spindle fibres of crane-fly spermatocytes andHaemanthus endosperm cells. Biochem Cell Biol 70: 664–676

    PubMed  Google Scholar 

  • — —, Bajer AS (1993) Ultraviolet microbeam irradiation of chromosomal spindle fibres inHaemanthus katherinae endosperm. I. Behaviour of the irradiated region. J Cell Sci 105: 571–578

    PubMed  Google Scholar 

  • Dietz R (1972) Anaphase behavior of inversions in living crane-fly spermatocytes. Chromosomes Today 3: 70–85

    Google Scholar 

  • Forer A (1965) Local reduction of spindle fibre birefringence in livingNephrotoma suturalis (Loew) spermatocytes induced by ultraviolet microbeam irradiation. J Cell Biol 25: 95–117

    PubMed  Google Scholar 

  • — (1966) Characterization of the mitotic traction system, and evidence that birefringent spindle fibres neither produce nor transmit force for chromosome movement. Chromosoma 19: 44–98

    PubMed  Google Scholar 

  • — (1982) Possible roles of cytoskeletal elements in mitosis. In: Lloyd CW (ed) The cytoskeleton in plant growth and development. Academic Press, London, pp 185–201

    Google Scholar 

  • — (1985) Does actin produce the force that moves a chromosome to the pole during anaphase? Can J Biochem Cell Biol 63: 585–598

    PubMed  Google Scholar 

  • — (1988) Do chromosomes chew their way to the pole or are they pulled by actin? J Cell Sci 91: 449–453

    PubMed  Google Scholar 

  • —, Jackson WT (1975) Actin in the higher plantHaemanthus katherinae Baker. Cytobiologie 10: 217–226

    Google Scholar 

  • — — (1976) Actin filaments in the endosperm mitotic spindles in a higher plant,Haemanthus katherinae Baker. Cytobiologie 12: 199–214

    Google Scholar 

  • — — (1979) Actin in spindles ofHaemanthus katherinae endosperm. I. General results using various glycerination methods. J Cell Sci 37: 323–347

    PubMed  Google Scholar 

  • — —, Engberg A (1979) Actin in spindles ofHaemanthus katherinae endosperm. II. Distribution of actin in chromosomal spindle fibres, determined by analysis of serial sections. J Cell Sci 37: 349–371

    PubMed  Google Scholar 

  • Garel JR, Job D, Margolis RL (1987) Model of anaphase chromosome movement based on polymer-guided diffusion. Proc Natl Acad Sci USA 84: 3599–3603

    PubMed  Google Scholar 

  • Gorbsky GJ (1992) Chromosome motion in mitosis. BioEssays 14: 73–80

    PubMed  Google Scholar 

  • —, Sammak PJ, Borisy GG (1987) Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol 104: 9–18

    PubMed  Google Scholar 

  • Gordon GW (1980) The control of chromosome motion: ultraviolet microbeam irradiation of kinetochore fibres. PhD Thesis, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Google Scholar 

  • Gruzdev AD (1972) Critical review of some hypotheses of anaphase chromosome movements. Tsitologiya 14: 141–149 (in Russian, English version from National Research Council of Canada, Ottawa, Ontario, Technical Translation Number 1758)

    Google Scholar 

  • Inoué S, Sato H (1967) Cell motility by labile association of microtubules. J Gen Physiol 50: 259–292

    PubMed  Google Scholar 

  • Lees-Miller JP, Helfman DM, Schroer TA (1992) A vertebrate actinrelated protein is a component of a multisubunit complex involved in microtubule-based motility. Nature 359: 244–246

    PubMed  Google Scholar 

  • Leslie RJ, Pickett-Heaps JD (1983) Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation. J Cell Biol 96: 548–561

    PubMed  Google Scholar 

  • — — (1984) Spindle microtubule dynamics following ultraviolet microbeam irradiations of mitotic diatoms. Cell 36: 717–727

    PubMed  Google Scholar 

  • Lloyd CW, Traas JA (1988) The role of F-actin in determining the division plane of carrot suspension cells. Drug studies. Development 102: 211–221

    Google Scholar 

  • Margolis RL, Wilson L, Kiefer BI (1978) Mitotic mechanism based on intrinsic microtubule behaviour. Nature 272: 450–452

    PubMed  Google Scholar 

  • McCurdy DW, Gunning ES (1990) Reorganization of cortical actin microfilaments and microtubules at preprophase and mitosis in wheat root-tip cells: a double label immunofluorescence study. Cell Motil Cytoskeleton 15: 76–87

    Google Scholar 

  • McIntosh JR, Cande WZ, Snyder JA (1975) Structure and physiology of the mammalian mitotic spindle. In: Inoué S, Stephens RE (eds) Molecules and cell movement. Raven, New York, pp 31–76

    Google Scholar 

  • —, Hepler PK, Van Wie DG (1969) Model for mitosis. Nature 224: 659–663

    Google Scholar 

  • Nicklas RB (1988) Chromosomes and kinetochores do more in mitosis than previously thought. In: Gustafson JP, Appels A, Kaufman RJ (eds) Chromosome structure and function: the impact of new concepts. Plenum, New York, pp 53–74

    Google Scholar 

  • —, Kubai DF, Hays TS (1982) Spindle microtubules and their mechanical associations after micromanipulation in anaphase. J Cell Biol 95: 91–104

    PubMed  Google Scholar 

  • Palevitz BA (1987) Accumulation of F-actin during cytokinesis inAllium. Correlation with microtubule distribution and the effects of drugs. Protoplasma 141: 24–32

    Google Scholar 

  • Panteris E, Apostolakos P, Galatis B (1992) The organization of Factin in root tip cells ofAdiantum capillus veneris throughout the cell cycle. A double label fluorescence microscopy study. Protoplasma 170: 128–137

    Google Scholar 

  • Pfarr CM, Coue M, Grissom PM, Hays TS, Porter ME, McIntosh JR (1990) Cytoplasmic dynein is localized to kinetochores during mitosis. Nature 345: 263–265

    PubMed  Google Scholar 

  • Sanger JW (1975) Presence of actin during chromosomal movement. Proc Natl Acad Sci USA 72: 2451–2455

    PubMed  Google Scholar 

  • Schmit AC, Lambert AM (1990) Microinjected fluorescent phalloidin in vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells. Plant Cell 2: 129–138

    PubMed  Google Scholar 

  • Seagull RW, Falconer MM, Weerdenburg CA (1987) Microfilaments: dynamic arrays in higher plant cells. J Cell Biol 104: 995–1004

    PubMed  Google Scholar 

  • Sillers PJ, Forer A (1983) Action spectrum for changes in spindle fibre birefringence after ultraviolet microbeam irradiations of single chromosomal spindle fibres in crane-fly spermatocytes. J Cell Sci 62: 1–25

    PubMed  Google Scholar 

  • Snyder JA, Armstrong L, Stonington OG, Spurck TP, Pickett-Heaps JD (1991) UV-microbeam irradiations of the spindle: spindle forces and structural analysis of lesions. Eur J Cell Biol 55: 122–132

    PubMed  Google Scholar 

  • Spurck TP, Stonington OG, Snyder JA, Pickett-Heaps JD, Bajer A, Molé-Bajer J (1990) UV-microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production. J Cell Biol 111: 1505–1518

    PubMed  Google Scholar 

  • Staiger CJ, Cande WZ (1991) Microfilament distribution in maize meiotic mutants correlates with microtubule organization. Plant Cell 3: 637–644

    PubMed  Google Scholar 

  • Steuer ER, Wordeman L, Schroer TA, Scheetz MP (1990) Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature 345: 266–268

    PubMed  Google Scholar 

  • Subirana JA (1968) Role of spindle microtubules in mitosis. J Theor Biol 20: 117–123

    PubMed  Google Scholar 

  • 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: 387–395

    PubMed  Google Scholar 

  • —, Burgain S, Devaulx RD (1989) The organization of the cytoskeleton during meiosis in eggplant [Solanum melongena (L.)]: microtubules and F-actin are both necessary for coordinated meiotic division. J Cell Biol 92: 541–550

    Google Scholar 

  • Van Lammeren AAM, Bednara J, Willemse MTM (1989) Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine phalloidin. Planta 178: 531–539

    Google Scholar 

  • Wilson P, Forer A (1987) Irradiations of rabbit myofibrils with an ultraviolet microbeam. I. Effects of ultraviolet light on the myofibril components necessary for contraction. Biochem Cell Biol 65: 363–375

    PubMed  Google Scholar 

  • — — (1988) Ultraviolet microbeam irradiation of chromosomal spindle fibres shears microtubules and permits study of the new free ends in vivo. J Cell Sci 91: 455–468

    PubMed  Google Scholar 

  • — — (1989) The behaviour of microtubules in chromosomal spindle fibres irradiated singly or doubly with ultraviolet light. J Cell Sci 94: 625–634

    PubMed  Google Scholar 

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Czaban, B.B., Forer, A. Rhodamine-phalloidin and anti-tubulin antibody staining of spindle fibres that were irradiated with an ultraviolet microbeam. Protoplasma 178, 18–27 (1994). https://doi.org/10.1007/BF01404117

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