, Volume 131, Issue 1, pp 60–74 | Cite as

Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

  • T. P. Spurck
  • J. D. Pickett-Heaps
  • M. W. Klymkowsky


To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK1 cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPγS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.

We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.


Mitosis ATP Metabolic inhibitors Spindle Microtubule 


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  1. Bershadsky, A. D., Gelfand, V. I., 1981: ATP-dependant regulation of cytoplasmic microtubule assembly. Proc. Nat. Acad. Sci. U.S.A.78, 3610–3613.Google Scholar
  2. — —,Svitkina, T. M., Tint, I. S., 1980: Destruction of microfilament bundles in mouse embryo fibroblasts treated with inhibitors of energy metabolism. Exp. Cell Res.127, 421–429.Google Scholar
  3. Bonne, D., Pantaloni, D., 1982: Mechanism of tubulin assembly: guanosine 5′-triphosphate hydrolysis decreases the rate of microtubule depolymerization. Biochem.21, 1075–1081.Google Scholar
  4. Cohn, S. A.,Tippit, D. H.,Spurck, T., 1985: Microtubule Dynamics in the spindle. A thermodynamic description. Submitted.Google Scholar
  5. De Brabander, M., de Mey, J., 1980: The microtubule nucleating and organizing activity of kinetochores and centrosomes in living cells. In: Microtubules and Microtubule Inhibitors 1980. (de Brabander, M., de Mey, J., eds.), pp. 255–268. Amsterdam: Elsevier/North-Holland Biomed. Press.Google Scholar
  6. —,Geuens, G., Nuydens, R., Willebrords, R., de Mey, J., 1981: Microtubule assembly in living cells after release from nocodazole block: the effects of metabolic inhibitors, taxol and pH. Cell Biol. Int. Rep.5, 913–920.Google Scholar
  7. — — — — —, 1982: Microtubule stability and assembly in living cells: the influence of metabolic inhibitors, taxol and pH. Cold Spring Harbor Symp. Quant. Biol.46, 227–240.Google Scholar
  8. Deery, W. J., Means, A. R., Brinkley, B. R., 1984: Calmodulin-microtubule association in cultured mammalin cells. J. Cell Biol.98, 904–910.Google Scholar
  9. 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.Google Scholar
  10. —, 1981: Light microscopic studies of chromosome movements in living cells. In: Mitosis/Cytokinesis (Zimmerman, A. M., Forer, A., eds.), pp. 135–154. New York: Academic Press.Google Scholar
  11. Frankel, F. R., 1976: Organization and energy-dependent growth of microtubules in cells. Proc. Nat. Acad. Sci. U.S.A.73, 2798–2802.Google Scholar
  12. Fuller, G. M., Brinkley, B. R., 1976: Structure and control of assembly of cytoplasmic microtubules in normal and transformed cells. J. supramol. Struct.5, 497–514.Google Scholar
  13. Hepler, P. K., 1980: Membranes in the mitotic apparatus of barley cells. J. Cell. Biol.86, 490–499.Google Scholar
  14. Hoebeke, J., van Nijen, G., de Brabander, M., 1976: Interaction of nocodazole (R 17934), a new anti-tumoral drug, with rat brain tubulin. Biochem. biophys. Res. Comm.69, 319–324.Google Scholar
  15. Inoue, S., 1952: The effect of colchicine on the microscopic and submicroscopic structure of the mitotic spindle. Exp. Cell Res. Suppl.2, 305–318.Google Scholar
  16. —,Fuseler, J., Salmon, E. D., Ellis, W., 1975: Functional organization of mitotic microtubules: physical chemistry of thein vivo equilibrium system. Biophys. J.15, 725–744.Google Scholar
  17. Inoue, S., Sato, H., 1967: Cell motility by labile association of molecules: the nature of the mitotic spindle fibres and their role in chromosome movement. J. gen. Physiol.50, 259–292.Google Scholar
  18. Kiehart, D. P., 1981: Studies on thein vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. J. Cell Biol.88, 604–617.Google Scholar
  19. Kumagi, H., Nishida, E., Sakai, H., 1979: Microtubule assembly in the presence of adenosine triphosphate. J. Biochem.85, 495–502.Google Scholar
  20. Lee, J. C., Field, D. J., Lee, L. Y. Y., 1980: Effects of nocodazole on structures of calf brain tubulin. Biochem.19, 6209–6215.Google Scholar
  21. Leslie, R. J., Pickett-Heaps, J. D., 1984: Spindle microtubule dynamics following ultraviolet microbeam irradiations of mitotic diatoms. Cell36, 717–727.Google Scholar
  22. Margolis, R. L., Wilson, L., Kiefer, B. I., 1978: Mitotic mechanism based on instrinsic microtubule behaviour. Nature (Lond.)272, 450–452.Google Scholar
  23. McIntosh, J. R., 1977: Mitosisin vitro: isolates and models of the mitotic apparatus. In: Mitosis Facts and Questions (Little, M., Pawletz, N., Petzelt, C., Postingl, H., Schroeter, D., Zimmermann, H.-P., eds.), pp. 167–184. Berlin-Heidelberg-New York: Springer.Google Scholar
  24. McKeithan, T. W.,Rosenbaum, J. L., 1984: The biochemistry of microtubules. In: Cell and Muscle Motility (Shay, W. J., ed.), Plenum Pub. Corp.5, 255–288.Google Scholar
  25. Moskalewski, S., Thyberg, J., Friberg, U., 1980: Cold and metabolic inhibitor effects on cytoplasmic microtubules and the Golgi complex in cultured rat epiphyseal chondrocytes. Cell Tissue Res.210, 403–415.Google Scholar
  26. Olmsted, J. B., Borisy, G. G., 1973: Characterization of microtubule assembly in porcine brain extracts by viscometry. Biochem.12, 4282–4289.Google Scholar
  27. Penningroth, S. M., Kirschner, M. M., 1977: Nucleotide binding and phosphorylation in microtubule assemblyin vitro. J. mol. Biol.115, 643–673.Google Scholar
  28. Pickett-Heaps, J. D., Spurck, T. P., 1982: Studies on kinetochore function in mitosis. II. The effects of metabolic inhibitors on mitosis in the diatomHantzschia amphioxys. Europ. J. Cell Biol.28, 83–91.Google Scholar
  29. -Tippit, D. H.,Cohn, S. A.,Spurck, T. P., 1985: Microtubule dynamics in the spindle. Theoretical aspects of assembly/disassemblyin vivo. J. theoretic. Biol. in press.Google Scholar
  30. — —,Porter, K. R., 1982: Rethinking mitosis. Cell29, 729–744.Google Scholar
  31. Salmon, E. D., 1982 a: Mitotic spindles isolated from sea urchin eggs EGTA lysis buffers. In: Methods in Cell Biology (Wilson, L., ed.), Vol. 25, pp. 70–105. New York: Academic Press.Google Scholar
  32. —, 1982 b: Calcium, spindle microtubule dynamics and chromosome movement. Cell. Diff.11, 353–355.Google Scholar
  33. —,Begg, D. A., 1980: Functional implications of cold-stable microtubules in kinetochore fibres of insect spermatocytes during anaphase. J. Cell Biol.85, 853–865.Google Scholar
  34. Schaap, C. J., Forer, A., 1984 a: Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. I. Description of the system and general results. J. Cell Sci.65, 21–40.Google Scholar
  35. — —, 1984 b: Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres. II. Crane-fly spermatocyte chromosomal spindle fibres are not temperaturelabile. J. Cell Sci.65, 41–60.Google Scholar
  36. Schliwa, M., van Blerkom, J., 1981: Structural interaction of cytoskeletal components. J. Cell Biol.90, 222–235.Google Scholar
  37. Snyder, J. A., 1981: Studies of mitotic events using lysed cell models. In: Mitosis/Cytokinesis (Zimmermann, A. M., Forer, A., eds.), pp. 301–336. New York: Academic Press.Google Scholar
  38. Tippit, D. H., Pickett-Heaps, J. D., Leslie, R. J., 1980: Cell division in two large pennate diatomsHantzschia andNitzschia. III. A new proposal for kinetochore function during prometaphase. J. Cell Sci.86, 402–416.Google Scholar
  39. Zabrecky, J. R., Cole, R. D., 1982: Effect of ATP on the kinetics of microtubule assembly. Proc. Nat. Acad. Sci. U.S.A.257, 4633–4638.Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • T. P. Spurck
    • 1
  • J. D. Pickett-Heaps
    • 1
  • M. W. Klymkowsky
    • 1
  1. 1.Department of Molecular, Cellular and Developmental BiologyUniversity of ColoradoBoulderUSA

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