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. 1976 Jul 1;70(1):75–85. doi: 10.1083/jcb.70.1.75

Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs

PMCID: PMC2109816  PMID: 945280

Abstract

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.

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Selected References

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  1. Aronson J., Inoué S. Reversal by light of the action of N-methyl N-desacetyl colchicine on mitosis. J Cell Biol. 1970 May;45(2):470–477. doi: 10.1083/jcb.45.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borisy G. G., Olmsted J. B., Marcum J. M., Allen C. Microtubule assembly in vitro. Fed Proc. 1974 Feb;33(2):167–174. [PubMed] [Google Scholar]
  3. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. J Cell Biol. 1967 Aug;34(2):525–533. doi: 10.1083/jcb.34.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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. 1967 Jul;19(1):1–18. doi: 10.1016/s0022-5320(67)80057-1. [DOI] [PubMed] [Google Scholar]
  5. Bryan J. B., Nagle B. W., Doenges K. H. Inhibition of tubulin assembly by RNA and other polyanions: evidence for a required protein. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3570–3574. doi: 10.1073/pnas.72.9.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen W. D., Rebhun L. I. An estimate of the amount of microtubule protein in the isolated mitotic apparatus. J Cell Sci. 1970 Jan;6(1):159–176. doi: 10.1242/jcs.6.1.159. [DOI] [PubMed] [Google Scholar]
  7. Fuseler J. W. Repetitive procurement of mature gametes from individual sea stars and sea urchins. J Cell Biol. 1973 Jun;57(3):879–881. doi: 10.1083/jcb.57.3.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goldman R. D., Rebhun L. I. The structure and some properties of the isolated mitotic apparatus. J Cell Sci. 1969 Jan;4(1):179–209. doi: 10.1242/jcs.4.1.179. [DOI] [PubMed] [Google Scholar]
  9. Heidemann S. R., Kirschner M. W. Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies. J Cell Biol. 1975 Oct;67(1):105–117. doi: 10.1083/jcb.67.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Inoué S., Borisy G. G., Kiehart D. P. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. J Cell Biol. 1974 Jul;62(1):175–184. doi: 10.1083/jcb.62.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inoué S., Fuseler J., Salmon E. D., Ellis G. W. Functional organization of mitotic microtubules. Physical chemistry of the in vivo equilibrium system. Biophys J. 1975 Jul;15(7):725–744. doi: 10.1016/S0006-3495(75)85850-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inoué S., Sato H. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J Gen Physiol. 1967 Jul;50(6 Suppl):259–292. [PMC free article] [PubMed] [Google Scholar]
  13. Jacobs M. Tubulin nucleotide reactions and their role in microtubule assembly and dissociation. Ann N Y Acad Sci. 1975 Jun 30;253:562–572. doi: 10.1111/j.1749-6632.1975.tb19229.x. [DOI] [PubMed] [Google Scholar]
  14. MANGAN J., MIKI-NOUMURA T., GROSS P. R. PROTEIN SYNTHESIS AND THE MITOTIC APPARATUS. Science. 1965 Mar 26;147(3665):1575–1578. doi: 10.1126/science.147.3665.1575. [DOI] [PubMed] [Google Scholar]
  15. 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. 1975 Oct;67(1):189–199. doi: 10.1083/jcb.67.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Raff R. A., Brandis J. W., Green L. H., Kaumeyer J. F., Raff E. C. Microtubule protein pools in early development. Ann N Y Acad Sci. 1975 Jun 30;253:304–317. doi: 10.1111/j.1749-6632.1975.tb19209.x. [DOI] [PubMed] [Google Scholar]
  17. Raff R. A., Greenhouse G., Gross K. W., Gross P. R. Synthesis and storage of microtubule proteins by sea urchin embryos. J Cell Biol. 1971 Aug;50(2):516–527. doi: 10.1083/jcb.50.2.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Raff R. A., Kaumeyer J. F. Soluble microtubule proteins of the sea urchin embryo: partial characterization of the proteins and behavior of the pool in early development. Dev Biol. 1973 Jun;32(2):309–320. doi: 10.1016/0012-1606(73)90243-1. [DOI] [PubMed] [Google Scholar]
  19. Rebhun L. I., Jemiolo D., Ivy N., Mellon M., Nath J. Regulation of the in vivo mitotic apparatus by glycols and metabolic inhibitors. Ann N Y Acad Sci. 1975 Jun 30;253:362–377. doi: 10.1111/j.1749-6632.1975.tb19214.x. [DOI] [PubMed] [Google Scholar]
  20. Rebhun L. I., Rosenbaum J., Lefebvre P., Smith G. Reversible restoration of the birefringence of cold-treated, isolated mitotic apparatus of surf clam eggs with chick brain tubulin. Nature. 1974 May 10;249(453):113–115. doi: 10.1038/249113a0. [DOI] [PubMed] [Google Scholar]
  21. Rebhun L. I., Sander G. Ultrastructure and birefringence of the isolated mitotic apparatus of marine eggs. J Cell Biol. 1967 Sep;34(3):859–883. doi: 10.1083/jcb.34.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Salmon E. D. Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length. J Cell Biol. 1975 Jun;65(3):603–614. doi: 10.1083/jcb.65.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salmon E. D. Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly. J Cell Biol. 1975 Jul;66(1):114–127. doi: 10.1083/jcb.66.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sato H., Ellis G. W., Inoué S. Microtubular origin of mitotic spindle form birefringence. Demonstration of the applicability of Wiener's equation. J Cell Biol. 1975 Dec;67(3):501–517. doi: 10.1083/jcb.67.3.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Snyder J. A., McIntosh J. R. Initiation and growth of microtubules from mitotic centers in lysed mammalian cells. J Cell Biol. 1975 Dec;67(3):744–760. doi: 10.1083/jcb.67.3.744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stephens R. E. A thermodynamic analysis of mitotic spindle equilibrium at active metaphase. J Cell Biol. 1973 Apr;57(1):133–147. doi: 10.1083/jcb.57.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. TAYLOR E. W. THE MECHANISM OF COLCHICINE INHIBITION OF MITOSIS. I. KINETICS OF INHIBITION AND THE BINDING OF H3-COLCHICINE. J Cell Biol. 1965 Apr;25:SUPPL–SUPPL:160. doi: 10.1083/jcb.25.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Telzer B. R., Moses M. J., Rosenbaum J. L. Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells. Proc Natl Acad Sci U S A. 1975 Oct;72(10):4023–4027. doi: 10.1073/pnas.72.10.4023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weisenberg R. C. Changes in the organization of tubulin during meiosis in the eggs of the surf clam, Spisula solidissima. J Cell Biol. 1972 Aug;54(2):266–278. doi: 10.1083/jcb.54.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weisenberg R. C., Rosenfeld A. C. In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs. J Cell Biol. 1975 Jan;64(1):146–158. doi: 10.1083/jcb.64.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weisenberg R. The role of nucleotides in microtubule assembly. Ann N Y Acad Sci. 1975 Jun 30;253:573–576. doi: 10.1111/j.1749-6632.1975.tb19230.x. [DOI] [PubMed] [Google Scholar]
  33. Wilson L., Bamburg J. R., Mizel S. B., Grisham L. M., Creswell K. M. Interaction of drugs with microtubule proteins. Fed Proc. 1974 Feb;33(2):158–166. [PubMed] [Google Scholar]

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