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. 1985 Sep 1;101(3):766–777. doi: 10.1083/jcb.101.3.766

Properties of the kinetochore in vitro. II. Microtubule capture and ATP- dependent translocation

PMCID: PMC2113737  PMID: 4030894

Abstract

We have studied the interaction of preformed microtubules (MTs) with the kinetochores of isolated chromosomes. This reaction, which we call MT capture, results in MTs becoming tightly bound to the kinetochore, with their ends capped against depolymerization. These observations, combined with MT dynamic instability, suggest a model for spindle morphogenesis. In addition, ATP appears to mobilize dynamic processes at captured MT ends. We used biotin-labeled MT seeds to follow assembly dynamics at the kinetochore. In the presence of ATP and unlabeled tubulin, labeled MT segments translocate away from the kinetochore by polymerization of subunits at the attached end. We have termed this reaction proximal assembly. Further studies demonstrated that translocation could be uncoupled from MT assembly. We suggest that the kinetochore contains an ATPase activity that walks along the MT lattice toward the plus end. This activity may be responsible for the movement of chromosomes away from the pole in prometaphase.

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Selected References

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  1. Abdella P. M., Smith P. K., Royer G. P. A new cleavable reagent for cross-linking and reversible immobilization of proteins. Biochem Biophys Res Commun. 1979 Apr 13;87(3):734–742. doi: 10.1016/0006-291x(79)92020-5. [DOI] [PubMed] [Google Scholar]
  2. Allen C., Borisy G. G. Structural polarity and directional growth of microtubules of Chlamydomonas flagella. J Mol Biol. 1974 Dec 5;90(2):381–402. doi: 10.1016/0022-2836(74)90381-7. [DOI] [PubMed] [Google Scholar]
  3. Bajer A. S. Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis. J Cell Biol. 1982 Apr;93(1):33–48. doi: 10.1083/jcb.93.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergen L. G., Kuriyama R., Borisy G. G. Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro. J Cell Biol. 1980 Jan;84(1):151–159. doi: 10.1083/jcb.84.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlier M. F., Hill T. L., Chen Y. Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study. Proc Natl Acad Sci U S A. 1984 Feb;81(3):771–775. doi: 10.1073/pnas.81.3.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Brabander M., Geuens G., De Mey J., Joniau M. Nucleated assembly of mitotic microtubules in living PTK2 cells after release from nocodazole treatment. Cell Motil. 1981;1(4):469–483. doi: 10.1002/cm.970010407. [DOI] [PubMed] [Google Scholar]
  7. Euteneuer U., McIntosh J. R. Structural polarity of kinetochore microtubules in PtK1 cells. J Cell Biol. 1981 May;89(2):338–345. doi: 10.1083/jcb.89.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans L., Mitchison T., Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985 Apr;100(4):1185–1191. doi: 10.1083/jcb.100.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. FORER A. LOCAL REDUCTION OF SPINDLE FIBER BIREFRINGENCE IN LIVING NEPHROTOMA SUTURALIS (LOEW) SPERMATOCYTES INDUCED BY ULTRAVIOLET MICROBEAM IRRADIATION. J Cell Biol. 1965 Apr;25:SUPPL–SUPPL117. doi: 10.1083/jcb.25.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gard D. L., Kirschner M. W. A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol. 1985 Mar;100(3):764–774. doi: 10.1083/jcb.100.3.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gundersen G. G., Kalnoski M. H., Bulinski J. C. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. 1984 Oct;38(3):779–789. doi: 10.1016/0092-8674(84)90273-3. [DOI] [PubMed] [Google Scholar]
  12. Heidemann S. R., McIntosh J. R. Visualization of the structural polarity of microtubules. Nature. 1980 Jul 31;286(5772):517–519. doi: 10.1038/286517a0. [DOI] [PubMed] [Google Scholar]
  13. Hill T. L. Diffusion frequency factors in some simple examples of transition-state rate theory. Proc Natl Acad Sci U S A. 1976 Mar;73(3):679–683. doi: 10.1073/pnas.73.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Johnson K. A., Porter M. E., Shimizu T. Mechanism of force production for microtubule-dependent movements. J Cell Biol. 1984 Jul;99(1 Pt 2):132s–136s. doi: 10.1083/jcb.99.1.132s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kirsch M., Yarbrough L. R. Assembly of tubulin with nucleotide analogs. J Biol Chem. 1981 Jan 10;256(1):106–111. [PubMed] [Google Scholar]
  17. Leslie R. J., Saxton W. M., Mitchison T. J., Neighbors B., Salmon E. D., McIntosh J. R. Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching. J Cell Biol. 1984 Dec;99(6):2146–2156. doi: 10.1083/jcb.99.6.2146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Margolis R. L., Wilson L., Keifer B. I. Mitotic mechanism based on intrinsic microtubule behaviour. Nature. 1978 Mar 30;272(5652):450–452. doi: 10.1038/272450a0. [DOI] [PubMed] [Google Scholar]
  19. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J Cell Biol. 1985 Sep;101(3):755–765. doi: 10.1083/jcb.101.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  21. Mitchison T., Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. doi: 10.1038/312232a0. [DOI] [PubMed] [Google Scholar]
  22. Molè-Bajer J. Fine structural studies of apolar mitosis. Chromosoma. 1969;26(4):427–448. doi: 10.1007/BF00326354. [DOI] [PubMed] [Google Scholar]
  23. Nicklas R. B., Brinkley B. R., Pepper D. A., Kubai D. F., Rickards G. K. Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes. J Cell Sci. 1979 Feb;35:87–104. doi: 10.1242/jcs.35.1.87. [DOI] [PubMed] [Google Scholar]
  24. Peterson J. B., Ris H. Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci. 1976 Nov;22(2):219–242. doi: 10.1242/jcs.22.2.219. [DOI] [PubMed] [Google Scholar]
  25. Pickett-Heaps J. D., Tippit D. H., Porter K. R. Rethinking mitosis. Cell. 1982 Jul;29(3):729–744. doi: 10.1016/0092-8674(82)90435-4. [DOI] [PubMed] [Google Scholar]
  26. Rieder C. L., Borisy G. G. The attachment of kinetochores to the pro-metaphase spindle in PtK1 cells. Recovery from low temperature treatment. Chromosoma. 1981;82(5):693–716. doi: 10.1007/BF00285776. [DOI] [PubMed] [Google Scholar]
  27. Rieder C. L. The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. Int Rev Cytol. 1982;79:1–58. doi: 10.1016/s0074-7696(08)61672-1. [DOI] [PubMed] [Google Scholar]
  28. Rieder C. L. The structure of the cold-stable kinetochore fiber in metaphase PtK1 cells. Chromosoma. 1981;84(1):145–158. doi: 10.1007/BF00293368. [DOI] [PubMed] [Google Scholar]
  29. Roos U. P. Light and electron microscopy of rat kangaroo cells in mitosis. III. Patterns of chromosome behavior during prometaphase. Chromosoma. 1976 Mar 10;54(4):363–385. doi: 10.1007/BF00292816. [DOI] [PubMed] [Google Scholar]
  30. Roos U. P. Mitosis in the cellular slime mold Polysphondylium violaceum. J Cell Biol. 1975 Feb;64(2):480–491. doi: 10.1083/jcb.64.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Salmon E. D., Begg D. A. Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase. J Cell Biol. 1980 Jun;85(3):853–865. doi: 10.1083/jcb.85.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Salmon E. D., Leslie R. J., Saxton W. M., Karow M. L., McIntosh J. R. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol. 1984 Dec;99(6):2165–2174. doi: 10.1083/jcb.99.6.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schibler M. J., Pickett-Heaps J. D. Mitosis in Oedogonium: spindle microfilaments and the origin of the kinetochore fiber. Eur J Cell Biol. 1980 Oct;22(2):687–698. [PubMed] [Google Scholar]
  35. Sheetz M. P., Chasan R., Spudich J. A. ATP-dependent movement of myosin in vitro: characterization of a quantitative assay. J Cell Biol. 1984 Nov;99(5):1867–1871. doi: 10.1083/jcb.99.5.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sluder G., Rieder C. L. Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes. J Cell Biol. 1985 Mar;100(3):897–903. doi: 10.1083/jcb.100.3.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon. Cell. 1985 Mar;40(3):559–569. doi: 10.1016/0092-8674(85)90204-1. [DOI] [PubMed] [Google Scholar]
  38. Webb B. C., Wilson L. Cold-stable microtubules from brain. Biochemistry. 1980 Apr 29;19(9):1993–2001. doi: 10.1021/bi00550a041. [DOI] [PubMed] [Google Scholar]
  39. Witt P. L., Ris H., Borisy G. G. Origin of kinetochore microtubules in Chinese hamster ovary cells. Chromosoma. 1980;81(3):483–505. doi: 10.1007/BF00368158. [DOI] [PubMed] [Google Scholar]
  40. Witt P. L., Ris H., Borisy G. G. Structure of kinetochore fibers: microtubule continuity and inter-microtubule bridges. Chromosoma. 1981;83(4):523–540. doi: 10.1007/BF00328277. [DOI] [PubMed] [Google Scholar]

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