Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Jan 1;110(1):81–95. doi: 10.1083/jcb.110.1.81

Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells

PMCID: PMC2115982  PMID: 2295685

Abstract

During mitosis in cultured newt pneumocytes, one or more chromosomes may become positioned well removed (greater than 50 microns) from the polar regions during early prometaphase. As a result, these chromosomes are delayed for up to 5 h in forming an attachment to the spindle. The spatial separation of these chromosomes from the polar microtubule- nucleating centers provides a unique opportunity to study the initial stages of kinetochore fiber formation in living cells. Time-lapse Nomarski-differential interference contrast videomicroscopic observations reveal that late-attaching chromosomes always move, upon attachment, into a single polar region (usually the one closest to the chromosome). During this attachment, the kinetochore region of the chromosome undergoes a variable number of transient poleward tugs that are followed, shortly thereafter, by rapid movement of the chromosome towards the pole. Anti-tubulin immunofluorescence and serial section EM reveal that the kinetochores and kinetochore regions of nonattached chromosomes lack associated microtubules. By contrast, these methods reveal that the attachment and subsequent poleward movement of a chromosome correlates with the association of a single long microtubule with one of the kinetochores of the chromosome. This microtubule traverses the entire distance between the spindle pole and the kinetochore and often extends well past the kinetochore. From these results, we conclude that the initial attachment of a chromosome to the newt pneumocyte spindle results from an interaction between a single polar-nucleated microtubule and one of the kinetochores on the chromosome. Once this association is established, the kinetochore is rapidly transported poleward along the surface of the microtubule by a mechanism that is not dependent on microtubule depolymerization. Our results further demonstrate that the motors for prometaphase chromosome movement must be either on the surface of the kinetochore (i.e., within the corona but not the plate), distributed along the surface of the kinetochore microtubules, or both.

Full Text

The Full Text of this article is available as a PDF (7.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Brinkley B. R., Cartwright J., Jr Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. Ann N Y Acad Sci. 1975 Jun 30;253:428–439. doi: 10.1111/j.1749-6632.1975.tb19218.x. [DOI] [PubMed] [Google Scholar]
  3. Cassimeris L., Inoué S., Salmon E. D. Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy. Cell Motil Cytoskeleton. 1988;10(1-2):185–196. doi: 10.1002/cm.970100123. [DOI] [PubMed] [Google Scholar]
  4. Cassimeris L., Pryer N. K., Salmon E. D. Real-time observations of microtubule dynamic instability in living cells. J Cell Biol. 1988 Dec;107(6 Pt 1):2223–2231. doi: 10.1083/jcb.107.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Church K., Lin H. P. Meiosis in Drosophila melanogaster. II. The prometaphase-I kinetochore microtubule bundle and kinetochore orientation in males. J Cell Biol. 1982 May;93(2):365–373. doi: 10.1083/jcb.93.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Church K., Nicklas R. B., Lin H. P. Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm. J Cell Biol. 1986 Dec;103(6 Pt 2):2765–2773. doi: 10.1083/jcb.103.6.2765. [DOI] [PMC free article] [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. Forer A. Do anaphase chromosomes chew their way to the pole or are they pulled by actin? J Cell Sci. 1988 Dec;91(Pt 4):449–453. doi: 10.1242/jcs.91.4.449. [DOI] [PubMed] [Google Scholar]
  9. Geuens G., Hill A. M., Levilliers N., Adoutte A., DeBrabander M. Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase. J Cell Biol. 1989 Mar;108(3):939–953. doi: 10.1083/jcb.108.3.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ghosh S., Paweletz N. Centrosome-kinetochore interaction in multinucleate cells. Chromosoma. 1987;95(2):136–143. doi: 10.1007/BF00332186. [DOI] [PubMed] [Google Scholar]
  11. Gibbons I. R. Dynein ATPases as microtubule motors. J Biol Chem. 1988 Nov 5;263(31):15837–15840. [PubMed] [Google Scholar]
  12. Goldstein L. S. Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster. Cell. 1981 Sep;25(3):591–602. doi: 10.1016/0092-8674(81)90167-7. [DOI] [PubMed] [Google Scholar]
  13. Gorbsky G. J., Sammak P. J., Borisy G. G. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol. 1987 Jan;104(1):9–18. doi: 10.1083/jcb.104.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hays T. S., Wise D., Salmon E. D. Traction force on a kinetochore at metaphase acts as a linear function of kinetochore fiber length. J Cell Biol. 1982 May;93(2):374–389. doi: 10.1083/jcb.93.2.374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hill T. L., Kirschner M. W. Bioenergetics and kinetics of microtubule and actin filament assembly-disassembly. Int Rev Cytol. 1982;78:1–125. [PubMed] [Google Scholar]
  16. Inoué S. Cell division and the mitotic spindle. J Cell Biol. 1981 Dec;91(3 Pt 2):131s–147s. doi: 10.1083/jcb.91.3.131s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Janicke M. A., LaFountain J. R., Jr Malorientation in half-bivalents at anaphase: analysis of autosomal laggards in untreated, cold-treated, and cold-recovering crane fly spermatocytes. J Cell Biol. 1984 Mar;98(3):859–869. doi: 10.1083/jcb.98.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koshland D. E., Mitchison T. J., Kirschner M. W. Polewards chromosome movement driven by microtubule depolymerization in vitro. Nature. 1988 Feb 11;331(6156):499–504. doi: 10.1038/331499a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Marko M., Leith A., Parsons D. Three-dimensional reconstruction of cells from serial sections and whole-cell mounts using multilevel contouring of stereo micrographs. J Electron Microsc Tech. 1988 Aug;9(4):395–411. doi: 10.1002/jemt.1060090406. [DOI] [PubMed] [Google Scholar]
  21. Mc2ntosh J. R., Cande Z., Snyder J., Vanderslice K. Studies on the mechanism of mitosis. Ann N Y Acad Sci. 1975 Jun 30;253:407–427. doi: 10.1111/j.1749-6632.1975.tb19217.x. [DOI] [PubMed] [Google Scholar]
  22. McIntosh J. R., Cande W. Z., Snyder J. A. Structure and physiology of the mammalian mitotic spindle. Soc Gen Physiol Ser. 1975;30:31–76. [PubMed] [Google Scholar]
  23. McNeill P. A., Berns M. W. Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells. J Cell Biol. 1981 Mar;88(3):543–553. doi: 10.1083/jcb.88.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. doi: 10.1083/jcb.101.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mitchison T. J. Microtubule dynamics and kinetochore function in mitosis. Annu Rev Cell Biol. 1988;4:527–549. doi: 10.1146/annurev.cb.04.110188.002523. [DOI] [PubMed] [Google Scholar]
  27. Mitchison T., Evans L., Schulze E., Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. doi: 10.1016/0092-8674(86)90283-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Nicklas R. B. Chromosome micromanipulation. II. Induced reorientation and the experimental control of segregation in meiosis. Chromosoma. 1967;21(1):17–50. doi: 10.1007/BF00330545. [DOI] [PubMed] [Google Scholar]
  31. Nicklas R. B., Kubai D. F. Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation. Chromosoma. 1985;92(4):313–324. doi: 10.1007/BF00329815. [DOI] [PubMed] [Google Scholar]
  32. Nicklas R. B. Measurements of the force produced by the mitotic spindle in anaphase. J Cell Biol. 1983 Aug;97(2):542–548. doi: 10.1083/jcb.97.2.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nicklas R. B. The forces that move chromosomes in mitosis. Annu Rev Biophys Biophys Chem. 1988;17:431–449. doi: 10.1146/annurev.bb.17.060188.002243. [DOI] [PubMed] [Google Scholar]
  34. Paschal B. M., Shpetner H. S., Vallee R. B. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. doi: 10.1083/jcb.105.3.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Paschal B. M., Vallee R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. doi: 10.1038/330181a0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Pickett-Heaps J. D., Tippit D. H. The diatom spindle in perspective. Cell. 1978 Jul;14(3):455–467. doi: 10.1016/0092-8674(78)90232-5. [DOI] [PubMed] [Google Scholar]
  38. Rickards G. K. Prophase chromosome movements in living house cricket spermatocytes and their relationship to prometaphase, anaphase and granule movements. Chromosoma. 1975;49(4):407–455. doi: 10.1007/BF00285133. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Rieder C. L., Davison E. A., Jensen L. C., Cassimeris L., Salmon E. D. Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spindle. J Cell Biol. 1986 Aug;103(2):581–591. doi: 10.1083/jcb.103.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Roos U. P. Light and electron microscopy of rat kangaroo cells in mitosis. II. Kinetochore structure and function. Chromosoma. 1973;41(2):195–220. doi: 10.1007/BF00319696. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Schliwa M., van Blerkom J. Structural interaction of cytoskeletal components. J Cell Biol. 1981 Jul;90(1):222–235. doi: 10.1083/jcb.90.1.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Steffen W., Fuge H., Dietz R., Bastmeyer M., Müller G. Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation? J Cell Biol. 1986 May;102(5):1679–1687. doi: 10.1083/jcb.102.5.1679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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. 1980 Aug;86(2):402–416. doi: 10.1083/jcb.86.2.402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wadsworth P., Shelden E., Rupp G., Rieder C. L. Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase. J Cell Biol. 1989 Nov;109(5):2257–2265. doi: 10.1083/jcb.109.5.2257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Walker R. A., Inoué S., Salmon E. D. Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro. J Cell Biol. 1989 Mar;108(3):931–937. doi: 10.1083/jcb.108.3.931. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES