Abstract
When late prophase PtK1 cells are chilled to 6 ° C the nuclear envelope (NE) breaks down as in normal cells but the spindle is inhibited from forming. When these cells are subsequently warmed to 18 ° C the spindle slowly forms and pro-metaphase congression ensues. Using this approach we have been able to experimentally eliminate the influence of asynchronous NE breakdown on the formation and development of the spindle, and also to slow down (and thus increase the temporal separation of) the subsequent events which occur during the initial stages of spindle formation. Correlative light and high voltage electron microscopic studies on these cells, fixed after various times of recovery, reveal the following results: 1) the centrosomes generate microtubules (MTs) well before MTs are seen to be associated with the kinetochores; 2) as in untreated PtK1 cells (Roos, 1973a, 1976) the order in which chromosomes attach to the forming spindle is influenced by their proximity to a centrosome-kinetochores closest to a centrosome appear stretched towards the centrosome at a time during recovery when other kinetochores, more distal to the centrosome appear unstretched and unoriented; 3) as in untreated cells (Heneen, 1970; Roos, 1976) the predominant behavior during recovery is for a chromosome to initially mono-orient and associate with the near centrosome and only later to develop a bipolar association; and 4) MTs associated with early pro-metaphase kinetochores are almost always oriented towards a centrosome. — From our results we conclude that the proximity effect and the tendency of pro-metaphase chromosomes in PtK1 to initially mono-orient and associate with the near centrosome cannot be ascribed, as suggested by Roos (1976), to influences arising during the asynchronous breakdown of the NE. Rather, our data clearly demonstrate that a kinetochore-centrosome interaction occurs during spindle formation which cannot be attributed to transient influences. The proximity effect and the predominant tendency of PtK1 pro-metaphase chromosomes to mono-orient to the near pole are taken to signify the existance of a centrosomal influence on the attachment and orientation of chromosomes. Two possible mechanisms for this influence, both involving a structural interaction between the centrosome and the kinetochore, are outlined.
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Aubin, J.E., Osborn, M., Weber, K.: Variations in the distribution and migration of centriolar duplexes in mitotic PtK2 cells studied by immunofluorescence microscopy. J. Cell Sci. 43, 177–194 (1980)
Bajer, A.S., Molè-Bajer, J.: Spindle dynamics and chromosome movement. Int. Rev. Cytol., Suppl. 2 (1972)
Barber, H., Callan, H.: The effects of cold and colchicine on mitosis in the newt. Proc. roy. Soc., Lond., B. 131, 258–274 (1942)
Behnke, O., Rostgaard, J.: Your “third hand” in mounting serial sections on grids for electron microscopy. Stain Technol. 39, 205–208 (1964)
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. Ultrastr. Res. 19, 1–18 (1967)
DeBrabander, M., Geuens, G., DeMey, J., Joniau, M.: Light microscopic and ultrastructural distribution of immunoreactive tubulin in mitotic mammalian cells. Biol. cellulaire 34, 213–226 (1979a)
DeBrabander, M., DeMey, J., Geuens, G., Joniau, M.: The distribution, function and regulation of microtubules in cultured cells studied with a new “complete” immunocytochemical approach. 37th Ann. Proc. Electron Microsc. Soc. Amer. (G.W. Bailey, ed), pp. 10–13 (1979b)
DeBrabander, M., Geuens, G., DeMey, J., Joniau, M.: The organized assembly and function of the microtubule system throughout the cell cycle. In: Cell movement and neoplasia (M. DeBrabander, M. Mareel and L. DeRidder, eds.), pp 19–40. New York: Pergamon Press (1980a)
DeBrabander, M., Geuens, G., Nuydens, R., Willebrords, R., DeMey, J.: The microtubule nucleating and organizing activity of kinetochores and centrosomes in living PtK2 cells. In: Microtubules and microtubule inhibitors (M. DeBrabander and J. DeMey, eds.), pp. 255–268. New York: North Holland 1980b
DeRobertis, E.M., Longthorne, R.R., Gurdon, J.B.: Intracellular migration of nuclear proteins in Xenopus oocytes. Nature (Lond) 272, 254–256 (1978)
Goode, D.: Kinetics of microtubule formation after cold disaggregation of the mitotic apparatus. J. molec. Biol. 80, 531–538 (1973)
Heneen, W.K.: In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells. Chromosoma (Berl.) 29, 88–117 (1970)
Heneen, W.K.: Ultrastructure of the prophase kinetochore in cultured cells of rat kangaroo (Potorous tridactylis). Hereditas (Lond) 79, 209–220 (1975)
Inoué, S.: Effect of temperature on the birefringence of the mitotic spindle. Biol. Bull 103, 316a (1952)
Inoué, S.: Organization and function of the mitotic spindle. In: Primitivve motile systems in cell biology (R.D. Allen and N. Kamiya, eds.). New York. Academic Press 1964
Izutsu, J., Sato, H., Nakabayashi, H., Aoki, N.: The behavior of spindle fibers and movement of chromosomes in dividing grasshopper spermatocytes. Cell Structure and Function 2, 119–133 (1977)
Jensen, C., Bajer, A.S.: Effects of dehydration on the microtubules of the mitotic spindle. Studies in vitro and with the electron microscope. J. Ultrastr. Res. 26, 367–386 (1969)
Jensen, C., Jensen, L., Rieder, C.L.: The occurrence and the structure of primary cilia in a subline of Potorous tridactylus (PtK1). Exp. Cell Res. 123, 444–449 (1979)
Jokelainen, P.T.: The differentiation of sister kinetochores during metakinesis. J. Cell Biol 27 (2 pt 2), 48a (1965)
Jokelainen, P.T.: The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells. J. Ultrastr. Res. 19, 19–44 (1967)
Lambert, A.M.: The role of chromosomes in anaphase trigger and nuclear envelope activity in spindle formation. Chromosoma (Berl.) 76, 295–308 (1980)
Lambert, A.M., Bajer, A.S.: Microtubule distribution and reversible arrest of chromosome movements induced by low temperature. Cytobiologie 15, 1–23 (1977)
Leibovitz, A.: The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Amer. J. Hygene 78, 173–180 (1963)
Mazia, D.: Mitosis and the physiology of cell division. In: The cell (J. Brachet and A.E. Mirsky, eds), pp 77–412. New York: Academic Press 1961
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, 189–199 (1975)
McIntosh, J.R., Cande, W.Z., Snyder, J.D.: Structure and physiology of the mammalian mitotic spindle. In: Molecules and cell movement (S. Inoué and R.E. Stephens, eds.), pp 31–74. New York: Raven Press 1975
Molè-Bajer, J.: The role of centrioles in the development of the astral spindle (newt). Cytobios 13, 17–40 (1975)
Molè-Bajer, J., Bajer, A.S.: Studies of selected endosperm cells with the light and electron microscope. The technique. Cellule 67, 257–265 (1968)
Molè-Bajer, J., Bajer, A., Owczarzak, A.: Chromosome movements in pro-metaphase and aster transport in the newt. Cytobios 13, 45–65 (1975)
Moore, M.J.: Removal of glass coverslips from cultures flat embedded in epoxy resins using hydroflouric acid. J. Microsc. 104, 205–207 (1975)
Nicklas, R.B., Brinkley, B.R., Pepper, D.A., Kubai, D., Rickards, G.K.: Electron microscopy of spermatocytes previously studied in life: Methods and some observations on micromanipulated chromosomes. J. Cell Science 35, 87–104 (1979)
Paweletz, N.: Elektronenmikroskopische Untersuchungen am frühen Stadien der Mitose bei HeLa Zellen. Cytobiologie 9, 368–390 (1974)
Rattner, J.B., Branch, A., Hamakalo, B.A.: Electron microscopy of whole mount metaphase chromosomes. Chromosoma (Berl.) 52, 329–338 (1975)
Rattner, J.B., Berns, M.W.: Distribution of microtubules during centriole separation in rat kangaroo (Potorous) cells. Cytobios 15, 37–43 (1976a)
Rattner J.B., Berns, M.W.: Centriole behavior in early mitosis of rat kangaroo cells (PtK2). Chromosoma (Berl.) 54, 387–395 (1976b)
Rieder, C.L.: Localization of ribonucleoprotein in the trilaminar kinetochore of PtK1. J. Ultrastr. Res. 66, 109–119 (1979)
Rieder, C.L.: The structure of the mammalian kinetochore and of the centrohelidian centroplast as revealed by Bernhard's staining of thick sections. In: Microtubules and microtubule inhibitors (M. DeBrabander and J. DeMey, eds.), pp. 311–324. New York: North Holland 1980
Rieder, C.L., Bajer, A.S.: Effect of elevated temperatures on spindle microtubules and chromosome movements in cultured newt lung cells. Cytobios 18, 201–234 (1977)
Ris, H.: The anaphase movement of chromosomes in spermatocytes of the grashopper. Biol. Bull. 96, 90–106 (1949)
Roos, U-P.: Light and electron microscopy of rat kangaroo cells in mitosis. I. Formation and breakdown of the mitotic apparatus. Chromosoma (Berl.) 40, 43–82 (1973a)
Roos, U-P.: Light and electron microscopy of rat kangaroo cells in mitosis. II. Kinetochore structure and function. Chromosoma (Berl.) 41, 195–220 (1973b)
Roos, U-P.: Light and electron microscopy of rat kangaroo cells in mitosis. III. Patterns of chromosome behavior during prometaphase. Chromosoma (Berl.) 54, 363–385 (1976)
Roos, U-P.: The fibrillar organization of the kinetochore and the kinetochore region of mammalian chromosomes. Cytobiologie 16, 82–90 (1977)
Roth, L.E.: Electron microscopy of mitosis in amebae. III. Cold and urea treatments: a basis for tests of direct effects of mitotic inhibitors on microtubule formation. J. Cell Biol. 34, 47–59 (1967)
Stephens, R.E.: Studies on the development of the sea urchin Strongylocentrotus droebachiensis. II. Regulation of mitotic spindle equilibrium by environmental temperature. Biol. Bull 142, 145–159 (1972)
Tippit, D.H., Pickett-Heaps, J.D., Leslie, R.: Cell division in two large pennate diatoms Hantzschia and Nitzschia. III. A new proposal for kinetochore function during prometaphase. J. Cell Biol. 86, 402–416 (1980)
Wilson, E.B.: The Cell in Development and Heredity. 3rd edit. New York: MacMillian 1925
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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Rieder, C.L., Borisy, G.G. The attachment of kinetochores to the pro-metaphase spindle in PtK1 cells. Chromosoma 82, 693–716 (1981). https://doi.org/10.1007/BF00285776
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DOI: https://doi.org/10.1007/BF00285776