Abstract
Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK1) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 Å thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Borisy, G.G., Olmstead, J.B.: Nucleated assembly of microtubules in porcine brain extracts. Science 117, 1196–1197 (1972)
Brinkley, B.R., Cartwright, J.: Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. Direct microtubule counts. J. Cell Biol. 50, 416–431 (1971)
Brinkley, B.R., Fuller, G.M., Highfield, D.P.: Studies of microtubules in dividing and nondividing mammalian cells using antibody to 6-S bovine brain tubulin. In: Microtubules and microtubule inhibitors (M. Borgers and M. de Brabander, eds.), pp. 297–312. Amsterdam: North-Holland Publ. Comp. 1975a
Brinkley, B.R., McGill, M., Mace, M.L.: Mammalian chromosome structure: ultrastructural aspects of specialized regions and chromosome aberrations. In: Radiation research (O.F. Nygaard, H.I. Adler and W.K. Sinclair, eds.), pp. 673–691. New York: Academic Press, Inc. 1975b
Brinkley, B.R., Murphy, P., Richardson, L.C.: Procedure for embedding in situ selected cells cultured in vitro. J. Cell Biol. 35, 279–283 (1967a)
Brinkley, B.R., Stubblefield, E.: Fine structure of the kinetochore of Chinese hamster cells in vitro. Chromosoma (Berl.) 19, 28–43 (1966)
Brinkley, B.R., Stubblefield, E.: Ultrastructure and interaction of the kinetochore and centriole in mitosis and meiosis. Advanc. Cell Biol. 1, 119–185 (1970)
Brinkley, B.R., Stubblefield, E., Hsu, T.C.: The effects of Colcemid inhibition and reversal on the fine structure of the mitotic apparatus of Chinese hamster cells in vitro. J. Ultrastruct. Res. 19, 1–18 (1967b)
Comings, D.E., Okada, T.A.: Fine structure of kinetochore in Indian muntjac. Exp. Cell Res. 67, 97–110 (1971)
Dales, S.: Concerning the universality of a microtubule antigen in animal cells. J. Cell Biol. 52, 748–754 (1972)
Forer, A.: Actin filaments and birefringent spindle fibers during chromosome movements. In: Cell motility (R. Goldman, T. Pollard, J. Rosenbaum, eds.), Vol. 3, pp. 1273–1293. Cold Spring Harbor Laboratory 1976
Fuller, G.M., Brinkley, B.R., Boughter, J.M.: Immunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin. Science 187, 948–950 (1975)
Fulton, C.: Centrioles. In: Origin and continuity of cell organelles (J. Reinert and H. Ursprung, eds.), pp. 170–221. Berlin-Heidelberg-New York: Springer 1971
Gawadi, N.: Actin in the mitotic spindle. Nature (Lond.) 234, 410 (1971)
Gould, R.R., Borisy, G.G.: Discrete particles at the centrosomes and kinetochores of Chinese hamster ovary cells. J. Cell Biol. 70, 43a (1976)
Graham, R.C., Karnovsky, M.J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291–302 (1966)
Harris, P.: Some observations concerning metakinesis in sea urchin eggs. J. Cell Biol. 25, 73–77 (1965)
Hinkley, R., Telser, A.: Heavy meromyosin-binding filaments in mitotic apparatus of mammalian cells. Exp. Cell Res. 86, 161–164 (1974)
Jokelainen, P.T.: The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells. J. Ultrastruct. Res. 19, 19–44 (1967)
Kirschner, M.W., Honig, L.S., Williams, R.C.: Quantitative electron microscopy of microtubule assembly in vitro. J. molec Biol. 99, 263–276 (1975)
Krahenbuhl, J.P., Jamieson, J.D.: Localization of intracellular antigens by immunoelectron microscopy. Int. Rev. exp. Path. 13, 1–53 (1974)
Krishan, A., Buck, R.C.: Structure of the mitotic spindle in L strain fibroblasts. J. Cell Biol. 24, 433–444 (1965)
McGill, M., Brinkley, B.R.: Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin. J. Cell Biol. 67, 188–199 (1975)
McIntosh, J.R., Cande, W.Z., Snyder, J.A.: Structure and physiology of the mammalian mitotic spindle. In: Molecules and cell movement (S. Inoué and R.E. Stephens, eds.), pp. 31–76. New York: Raven Press 1975
Nicklas, R.B.: Mitosis. Advanc. Cell Biol. 2, 225–297 (1971)
Olmstead, J.B., Marcum, J.M., Johnson, K.A., Allen, C., Borisy, G.G.: Microtubule assembly: some possible regulatory mechanisms. J. Supramolec. Struct. 2, 429–450 (1974)
Rattner, J.B., Phillips, S.: Independence of centriole formation and DNA synthesis. J. Cell Biol. 57, 359–372 (1973)
Roos, U.P.: Light and electron microscopy of rat kangaroo cells in mitosis II. Kinetochore structure and function. Chromosoma (Berl.) 41, 195–220 (1973)
Sanger, J.W.: Presence of actin during chromosomal movement. Proc. nat. Acad. Sci. (Wash.) 72, 2451–2455 (1975)
Schneider, B.G.: Pericentriolar virus-like particles in mammalian cells: segregation of particles to daughter cells at mitosis. M.A. Thesis. Univ. of Texas Medical Branch, Galveston, Texas USA (1975)
Snyder, J.A., McIntosh, J.R.: Studies on the centriolar region of mammalian cells at the onset of mitosis. J. Cell Biol. 70, 368a (1976)
Stubblefield, E., Brinkley, B.R.: Architecture and function of the mammalian centriole. In: The origin and fate of cell organelles (K.B. Warren, ed.), pp. 175–218. New York: Academic Press, Inc. 1967
Telzer, B.R., Moses, M.J., Rosenbaum, J.L.: Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells. Proc. nat. Acad. Sci. (Wash.) 72, 4023–4027 (1975)
Weber, K., Bibring, T., Osborn, M.: Specific visualization of tubulin containing structures in tissue culture cells by immunofluorescence. Exp. Cell Res. 95, 111–120 (1975)
Weisenberg, R.C., Borisy, G.G., Taylor, E.W.: The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry 7, 4466–4478 (1968)
Wheatley, D.N.: Pericentriolar virus-like particles in Chinese hamster ovary cells. J. gen. Virol. 24, 395–399 (1974)
Wilson, L., Bryan, J.: Biochemical and pharmacological properties of microtubules. In: Advances in cell and molecular biology (C.J. DuPraw, ed.), Vol. 3, pp. 21–72. New York: Academic Press, Inc. 1974
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pepper, D.A., Brinkley, B.R. Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy. Chromosoma 60, 223–235 (1977). https://doi.org/10.1007/BF00329772
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00329772