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An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis

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Summary

Tubulin can be isolated and purified from Xenopus laevis egges through modification of Olmstedt's (1970) tubulin isolation method, viz. by repeating the vinblastin precipitation step after resuspension of the sediment in a detergent-containing stabilizing medium. By this we overcome the deleterious influence of the yolk granules in the isolation procedure. From 1 l of Xenopus laevis eggs 25 mg VB-paracrystals can be obtained. The apparent molecular weight of the purified tubulin is 52,800. Antiserum against the purified Xenopus VB-paracrystals, raised in 2 Chinchilla rabbits, cross-reacts in immunodiffusion tests in agar gels with rat brain tubulin and with tubulin isolated from Xenopus laevis eggs by the described procedure. Specific indirect fluorescence staining and appropriate control reactions reveal that cilia of Tetrahymena pyriformis, cytoplasmic networks in cultured mouse Leydig cells, as well as mitotic spindles and nuclear regions in paraffin sections of Xenopus laevis blastulae, react with the antibodies against Xenopus laevis egg tubulin as well as with monoclonal antibodies against pig brain tubulin.

These results provide additional evidence for the view that tubulin antibodies are neither species nor tissue specific and show that under appropriate conditions tubulin containing structures can be visualized in paraffin sections.

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References

  • Bluemink JG (1970) The first cleavage of the amphibian egg. An electron microscope study of the onset of cytokinesis in the egg of Ambystoma mexicanum. J Ultrastruct Res 32:142–166

    Google Scholar 

  • Bluemink JG (1972) Cortical wound healing in the amphibian egg: an electron microscopical study. J Ultrastruct Res 41:95–114

    Google Scholar 

  • Bluemink JG, Tertoolen LGJ (1978) The plasma membrane IMP pattern as related to animal/vegetal polarity in the amphibian egg. Dev Biol 62:334–343

    Google Scholar 

  • Bryan J (1971) Vinblastine and microtubules. I. Induction and isolation of crystals from sea urchin oocytes. Exp Cell Res 66:129–136

    Google Scholar 

  • Burgess DR, Schroeder TE (1979) The cytoskeleton and cytomusculature in embryogenesis — an overview. Methods Achiev Exp Pathol 8:171–189

    Google Scholar 

  • Campanella C, Gabbiani G (1980) Cytoskeletal and contractile proteins in coelomic oocytes, infertilized and fertilized eggs of Discoglossus pictus (Anura) Gamete Res 3:99–114

    Google Scholar 

  • Conolly JA, Kalnins VI (1978) Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera. J Cell Biol 79:526–532

    Google Scholar 

  • Dales S (1972) Concerning the universality of a microtubule antigen in animal cells. J Cell Biol 52:748–754

    Google Scholar 

  • De Brabander M, de Mey J, Joniau M, Geuens S (1977) Immunocytochemical visualization of microtubules and tubulin at the light- and electron-microscopic level. J Cell Sci 28:283–301

    Google Scholar 

  • Dumont JN, Wallace RA (1972) The effects of vinblastine on isolated Xenopus oocytes. J Cell Biol 53:605–610

    Google Scholar 

  • Dustin P (1978) Microtubules. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Dustin P (1980) Microtubules. Sci Am 243:59–69

    Google Scholar 

  • Elinson RP, Manes ME (1978) Morphology of the site of sperm entry on the frog egg. Dev Biol 63:67–75

    Google Scholar 

  • Franke WW, Rathke PC, Seib E (1976) Distribution and mode of arrangement of microfilamentous structures and actin in the cortex of the amphibian oocyte. Cytobiology 14:111–130

    Google Scholar 

  • Fujiwara K, Sato H (1972) VB-crystal formation in sea urchin eggs by short term VB-col incubation. J Cell Biol 55/2, part 2:79a

    Google Scholar 

  • Gerhart J, Ubbels G, Black S, Hara K, Kirschner M (1981) A reinvestigation of the role of the grey crescent in axis formation in Xenopus laevis. Nature 292:511–516

    Google Scholar 

  • Grasset E, Pongratz E, Brechbühler T (1956) Analyse immunochimique des constituants des venins de serpents par la méthode de précipitation en milieu gélifié. Ann Inst Pasteur 91:162–186

    Google Scholar 

  • Gröschel-Stewart U (1980) Immunocytochemistry of cytoplasmic contractile proteins. Int Rev Cytol 65:193–254

    Google Scholar 

  • Hara K, Tydeman P, Kirschner M (1980) A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. Proc Natl Acad Sci USA 77:462–466

    Google Scholar 

  • Harris P, Osborn M, Weber K (1980) Distribution of tubulin containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage. J Cell Biol 84:668–679

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Google Scholar 

  • Lazarides E (1980) Intermediate filaments as mechanical integrators of cellular space. Nature 283:249–256

    Google Scholar 

  • Manes ME, Barbieri FD (1977) On the possibility of spermaster involvement in dorso-ventral polarization and pronuclear migration in the amphibian egg. J Embryol Exp Morphol 40:187–197

    Google Scholar 

  • Manes ME, Elinson RP, Barbieri FD (1978) Formation of the amphibian grey crescent: effects of colchicine and cytochalasin B. Wilhelm Roux Arch Dev Biol 185:99–104

    Google Scholar 

  • Nagayama A, Dales S (1970) Rapid purification and immunological specificity of mammalian microtubulan paracrystals possessing in ATP-ase activity. Proc Natl Acad Sci USA 66:464–471

    Google Scholar 

  • Nieuwkoop PD, Faber J (1967) Normal table of Xenopus laevis (Daudin). North-Holland, Amsterdam

    Google Scholar 

  • Olmstedt JB, Carlson K, Klebe R, Ruddle F, Rosenbaum J (1970) Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proc Natl Acad Sci USA 65:129–136

    Google Scholar 

  • Ouchterloni O (1949) Antigen-antibody reactions in gels. Ark Kemi, Mineral Geol 26:1–9

    Google Scholar 

  • Pestell RQW (1975) Microtubular protein synthesis during oogenesis and early embryogenesis in Xenopus laevis. Biochem J 145:527–534

    Google Scholar 

  • Porter KR, Tucker JB (1981) The ground substance of the living cell. Sci Am 244:40–51

    Google Scholar 

  • Raff EC (1977) Microtubule proteins in axolotl eggs and developing embryos. Dev Biol 58:56–75

    Google Scholar 

  • Raff EC (1979) The control of microtubule assembly in vivo. Int Rev Cytol 59:1–96

    Google Scholar 

  • Sato H, Ohnuki Y, Fujiwara K (1976) Immunofluorescent anti-tubulin staining of spindle microtubules and critique for the technique. In: Goldman R, Pollard T, Rosenbaum J (eds) Cell motility. Cold Spring Harbor Laboratory, pp 419–436

  • Schatten G, Schatten H (1981) Effects of motility inhibitors during sea urchin fertilization. Exp Cell Res 135:311–330

    Google Scholar 

  • Schliwa M, Euteneuer U, Bulinski JC, Izant JG (1981) Calcium lability of cytoplasmic microtubules and its modulation by microtubule associated proteins. Proc Natl Acad Sci USA 78:1037–1041

    Google Scholar 

  • Shapiro AL, Vinuela M, Maizel JV (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun 28:815–820

    Google Scholar 

  • Shelanski ML, Gaskin F, Cantor CR (1973) Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci USA 70:765–768

    Google Scholar 

  • Sloboda RD (1980) The role of microtubules in cell structure and cell division. Am Sci 68:290–298

    Google Scholar 

  • Stearns ME, Brown DL (1979) Purification of cytoplasmic tubulin and microtubule organizing center proteins functioning in microtubule initiation from the alga Polytomella. Proc Natl Acad Sci USA 76:5745–5749

    Google Scholar 

  • Ubbels GA, Bleumink ARJ (1978) Microtubules in the dorsal yolk-free cytoplasm of the fertilized egg of Xenopus laevis (60 min p.f.). Abstr. XIII Int. Embryological Conference Berlin, 25–29 September, no. 29

  • Ubbels GA, Koster CH (1980) Egg rotation experiments in Xenopus laevis with special reference to the determination of dorso-ventrality. Poster XIV Intern. Embryological Conference Patras, Greece, 11–17 September

  • Ubbels GA, Hara K, Koster CH, Kirschner, MW (1982) Evidence for a functional role of the cytoskeleton in determination of the dorso-ventral axis in Xenopus laevis eggs. (Submitted to the J Embryol Exp Morphol)

  • Vacquier VD (1981) Dynamic changes of the egg cortex. Dev Biol 84:1–26

    Google Scholar 

  • Viklický V, Dráber P, Hašek J, Bártek J (1982) Production and characterization of a monoclonal antitubulin antibody. Cell Biol Int Rep 6:725–731

    Google Scholar 

  • Weatherbee JA (1981) Membranes and cell movement: Interactions of membranes with the proteins of the cytoskeleton. Int Rev Cytol [Suppl 12]:113–176

    Google Scholar 

  • Weissenberg RC (1972) Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177:1104–1105

    Google Scholar 

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Authors and Affiliations

  1. Department of Experimental Zoology, Charles University, Vinična 7, 2, Praha, Czechoslovakia

    J. Paleček & J. Mácha

  2. Hubrecht Laboratory, International Embryological Institute, Uppsalalaan 8, NL-3584 CT, Utrecht, The Netherlands

    G. A. Ubbels

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  1. J. Paleček
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  2. G. A. Ubbels
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  3. J. Mácha
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Paleček, J., Ubbels, G.A. & Mácha, J. An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis . Histochemistry 76, 527–538 (1982). https://doi.org/10.1007/BF00489907

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