Abstract
To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 μm sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean∼90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) and extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubles terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30–40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, and may have a functional role as well.
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References
Amos, L.A.: Structure of microtubules. In: Microtubules. (K. Roberts and J.S. Hyams, eds.), pp. 2–64. London: Academic Press 1979
Bajer, A., Molè-Bajer, J.: Formation of spindle fibers, kinetochore orientation and behavior of the nuclear envelope during mitosis in endosperm. Fine structural and in vitro studies. Chromosoma (Berl.) 27, 448–484 (1969)
Bajer, A., Molè-Bajer, J.: Spindle dynamics and chromosome movements. Int. Rev. Cytol. Suppl. 3, 1–271 (1972)
Bergen, L.G., Borisy, G.G.: Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J. Cell Biol. 84, 141–150 (1980)
Bergen, L.G., Kuriyama, R., Borisy, G.G.: Polarity of microtubules nucleated by Chinese hamster ovary cells in vitro. J. Cell Biol. 84, 151–159 (1980)
Borisy, G.G.: Polarity of microtubules in the mitotic spindle. J. molec. Biol. 124, 565–570 (1978)
Brinkley, B.R., Cartwright, J.C., Jr.: Ultrastructural analysis of mitotic spindle elongation in mammalian cells in vitro. J. Cell Biol. 50, 416–431 (1971)
Brinkley, B.R., Cartwright, J., Jr.: Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. New York Acad. Sci. 253, 428–439 (1975)
Brinkley, B.R., Fistel, S.H., Marcum, J.M., Pardue, R.L.: Microtubules in cultured cells: indirect immunofluorescent staining with tubulin antibody. Int. Rev. Cytol. 63, 59–95 (1980)
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 ovary cells. J. Ultrastruct. Res. 19, 1–18 (1967)
Dietrich, J.: Reconstructions tridimensionnelles de l'appareil mitotique à partir de coupes sériées longitudinales de méiocytes polliniques. Biol. Cellulaire 34, 77–82 (1979)
Euteneuer, U., McIntosh, J.R.: Polarity of midbody and phragmoplast microtubules. J. Cell Biol. 87, 509–515 (1980)
Forer, A., Jackson, W.T., Engberg, A.: Actin in spindles of Haemanthus katherinae endosperm. J. Cell Sci. 37, 349–371 (1979)
Fuge, H.: The arrangement of microtubules and the attachment of chromosomes to the spindle during anaphase in tipulid spermatocytes. Chromosoma (Berl.) 45, 245–260 (1974)
Fuge, H.: Ultrastructure of the mitotic spindle. Int. Rev. Cytol. Suppl. 6, 1–58 (1977a)
Fuge, H.: Ultrastructure of mitotic cells. In: Mitosis: Facts and questions. (M. Little, N. Paweletz, C. Petzelt, H. Ponstingl, D. Schroeter, H.-P. Zimmermann, eds.) pp. 51–68. Berlin, Heidelberg, New York: Springer (1977b)
Fuge, H.: Microtubule disorientation in anaphase half-spindles during autosome segregation in crane fly spermatocytes. Chromosoma (Berl.) 76, 309–328 (1980)
Fuge, H., Muller, W.: Mikrotubuli-Kontakt an Anaphasechromosomen in der I. meiotischen Teilung. Exp. Cell Res. 71, 241–245 (1972)
Galavazi, G., Schenk, H., Bootsma, D.: Synchronization of mammalian cells in vitro by inhibition of the DNA synthesis. Exp. Cell Res. 41, 428–451 (1965)
Gould, R.R., Borisy, G.G.: The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation. J. Cell Biol. 73, 601–615 (1977)
Heine, U.I., Kramarsky, B., Wendel, E., Suskind, R.G.: Enhanced proliferation of endogenous virus in Chinese hamster cells associated with microtubules and the mitotic apparatus of the host cell. J. gen. Virol. 44, 44–55 (1979)
Heneen, W.K.: Kinetochores and microtubules in multipolar mitosis and chromosome orientation. Exp. Cell Res. 91, 57–62 (1975)
Hepler, P.K., McIntosh, J.R., Cleland, S.: Intermicrotubule bridges in the mitotic spindle apparatus. J. Cell Biol. 45, 438–444 (1970)
Hughes-Schrader, S.: Reproduction in Acroschismus wheeleri Pierce. J. Morph. and Physiol. 39, 157–205 (1924)
Inoué, S.: Organization and function of the mitotic spindle. In: Primitive Motile Systems in Cell Biology (R.D. Allen, N. Kamiya, eds.), pp. 549–594. New York: Academic Press 1964
Inoué, S., Ritter, H., Jr.: Mitosis in Barbulanympha. II. Dynamics of a two-stage anaphase, nuclear morphogenesis and cytokinesis. J. Cell Biol. 77, 655–684 (1975)
Jensen, C., Bajer, A.: Spindle dynamics and arrangement of microtubules. Chromosoma (Berl.) 44, 73–89 (1973a)
Jensen, C., Bajer, A.: Kinetochore microtubules of Haemanthus endosperm during mitosis. J. Cell Biol. 59, 156a (1973b)
LaFountain, J.R., Jr., Davidson, L.A.: An analysis of spindle ultrastructure during prometaphase and metaphase of micronuclear division in Tetrahymena. Chromosoma (Berl.) 75, 293–308 (1979)
Lambert, A.M., Bajer, A.S.: Dynamics of spindle fibers and microtubules during anaphase and phragmoplast formation. Chromosoma (Berl.) 39, 101–144 (1972)
Lambert, A.M., Bajer, A.: Fine structure dynamics of the prometaphase spindle. J. Microsc. Biol. Cell. 23, 181–194 (1975)
Lambert, A.M., Bajer, A.: Microtubule distribution and reversible arrest of chromosome movements induced by low temperature. Cytobiol. 15, 1–23 (1977)
McDonald, K.L., Edwards, M.K., McIntosh, J.R.: Cross-sectional structure of the central mitotic spindle of Diatoma vulgare. J. Cell Biol. 83, 443–461 (1979)
McIntosh, J.R.: Bridges between microtubules. J. Cell Biol. 61, 166–187 (1974)
McIntosh, J.R.: Cell Division. In: Microtubules (K. Roberts and J.S. Hyams, eds.), pp. 382–441. London: Academic Press 1979
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 1975a
McIntosh, J.R., Cande, W.Z., Snyder, J.A., Vanderslice, J.: Studies on the mechanism of mitosis: In: The Biology of cytoplasmic microtubules (D. Soifer, ed.), pp. 407–427. New York: New York Academy of Sciences (1975b)
Nicklas, R.B.: Mitosis. In: Advances in cell biology (D.M. Prescott, L. Goldstein and E. McConkey, eds.), vol. 2, pp. 225–297. New York: Appleton-Century-Crofts 1971
Paweletz, N.: Electronenmikroscopische Untersuchungen an frühen Stadien der Mitose bei HeLa-Zellen. Cytobiol. 9, 368–390 (1974)
Rieder, C., Bajer, A.: Heat-induced reversible hexagonal packing of spindle microtubules. J. Cell Biol. 74, 717–725 (1977)
Rieder, C.L., Borisy, G.G.: The attachment of kinetochores to the pro-metaphase spindle in PtKl cells. Recovery from low temperature treatment. Chromosoma (Berl.) 82, 693–716 (1981)
Ris, H., Witt, P.L.: Structure of the mammalian kinetochore. Chromosoma (Berl.) 82, 153–170 (1981)
Tilney, L.B.: How microtubule patterns are generated: the relative importance of nucleation and bridging of microtubules in the formation of the axoneme of Raphidiophrys. J. Cell Biol. 51, 837–854 (1971)
Tucker, J.B.: Spatial organization of microtubules. In: Microtubules (K. Roberts and J.S. Hyams, eds.), pp. 315–357, London: Academic Press 1979
Wheatley, D.N.: Pericentriolar virus-like particles in Chinese hamster ovary cells. J. gen. Virol. 24, 395–399 (1974)
Wilson, H.J.: Arms and bridges on microtubules in the mitotic apparatus. J. Cell Biol. 40, 854–859 (1969)
Witt, P.L., Ris, H., Borisy, G.G.: Origin of kinetochore microtubules in Chinese hamster ovary cells. Chromosoma (Berl.) 81, 483–505 (1980)
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Witt, P.L., Ris, H. & Borisy, G.G. Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges. Chromosoma 83, 523–540 (1981). https://doi.org/10.1007/BF00328277
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DOI: https://doi.org/10.1007/BF00328277