Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos

Nature Cell Biology volume 2, pages 922–930 (2000)Cite this article

Abstract

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Injection of p50 dynamitin causes a gradient of effects that may represent a range of activities for cytoplasmic dynein during mitosis.
Figure 2: Anti-DHC immunostains distinct sites on chromosomes.
Figure 3: Inhibition of dynein perturbs the ability of kinetochores to properly align at the spindle equatorial plate during metaphase.
Figure 4: Inhibition of dynein activity disrupts the ability of kinetochores to move polewards during anaphase A.
Figure 5: Inhibition of dynein activity severely inhibits sister-chromatid separation during anaphase A.
Figure 6: Visualization of chromatid-to-pole movement on individual spindles during anaphase in control and p50-injected embryos.
Figure 7: Rates of poleward kinetochore and chromosome movements during anaphase in control and dynein-inhibited embryos.

Similar content being viewed by others

References

  1. Paschal, B. M. & Vallee, R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature 330, 181–183 (1987).

    Article  CAS  Google Scholar 

  2. Karki, S. & Holzbaur, E. L. Cytoplasmic dynein and dynactin in cell division and intracellular transport. Curr. Opin. Cell Biol. 11, 45–53 (1999).

    Article  CAS  Google Scholar 

  3. Sharp, D. J. et al. Functional coordination of three mitotic motors in drosophila embryos. Mol. Biol. Cell 11, 241– 253 (2000).

    Article  CAS  Google Scholar 

  4. Saunders, W. S., Koshland, D., Eshel, D., Gibbons, I. R. & Hoyt, M. A. Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation. J. Cell Biol. 128, 617– 624 (1995).

    Article  CAS  Google Scholar 

  5. Pfarr, C. M. et al. Cytoplasmic dynein is localized to kinetochores during mitosis . Nature 345, 263–265 (1990).

    Article  CAS  Google Scholar 

  6. Steuer, E. R., Wordeman, L., Schroer, T. A. & Sheetz, M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature 345, 266–268 ( 1990).

    Article  CAS  Google Scholar 

  7. Wordeman, L., Steuer, E. R., Sheetz, M. P. & Mitchison, T. Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes. J. Cell Biol. 114, 285– 294 (1991).

    Article  CAS  Google Scholar 

  8. Echeverri, C. J., Paschal, B. M., Vaughan, K. T. & Vallee, R. B. Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis. J. Cell Biol. 132, 617– 633 (1996).

    Article  CAS  Google Scholar 

  9. Starr, D. A., Williams, B. C., Hays, T. S. & Goldberg, M. L. Zw10 helps recruit dynactin and dynein to the kinetochore. J. Cell Biol. 142, 763–774 ( 1998).

    Article  CAS  Google Scholar 

  10. Karki, S., LaMonte, B. & Holzbaur, E. L. Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells. J. Cell Biol. 142, 1023– 1034 (1998) [published erratum J. Cell Biol. 143, 560; 1998].

    Article  CAS  Google Scholar 

  11. McNeill, P. A. & Berns, M. W. Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells . J. Cell Biol. 88, 543– 553 (1981).

    Article  CAS  Google Scholar 

  12. Rieder, C. L. & Salmon, E. D. Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J. Cell Biol. 124, 223– 233 (1994).

    Article  CAS  Google Scholar 

  13. Bowman, A. B. et al. Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis. J. Cell Biol. 146, 165–180 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Karess, R. E. & Glover, D.M. rough deal: a gene required for proper mitotic segregation in Drosophila. J. Cell Biol. 109, 2951–2961 (1989).

    Article  CAS  Google Scholar 

  15. Williams, B. C., Karr, T. L., Montgomery, J. M. & Goldberg, M. L. The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset. J. Cell Biol. 118, 759– 773 (1992).

    Article  CAS  Google Scholar 

  16. Williams, B. C., Gatti, M. & Goldberg, M. L. Bipolar spindle attachments affect redistributions of Zw10, a Drosophila centromere/kinetochore component required for accurate chromosome segregation. J. Cell Biol. 134, 1127–1140 (1996).

    Article  CAS  Google Scholar 

  17. Yucel, J. K. et al. CENP-meta, an essential kinetochore kinesin required for the maintenance of metaphase chromosome alignment in Drosophila. J. Cell Biol. 150, 1–12 (2000).

    Article  CAS  Google Scholar 

  18. Moore, D. P., Page, A. W., Tang, T. T., Kerrebrock, A. W. & Orr-Weaver, T. L. The cohesion protein MEI-S332 localizes to condensed meiotic and mitotic centromeres until sister chromatids separate. J. Cell Biol. 140, 1003–1012 (1998).

    Article  CAS  Google Scholar 

  19. Sullivan, W. & Theurkauf, W. E. The cytoskeleton and morphogenesis of the early Drosophila embryo. Curr. Opin. Cell Biol. 7, 18–22 (1995).

    Article  CAS  Google Scholar 

  20. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996).

    Article  CAS  Google Scholar 

  21. Robinson, J. T., Wojcik, E. J., Sanders, M. A., McGrail, M. & Hays, T. S. Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila. J. Cell Biol. 146, 597–608 (1999).

    Article  CAS  Google Scholar 

  22. Waters, J. C., Mitchison, T. J., Rieder, C. L. & Salmon, E. D. The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work. Mol. Biol. Cell 7, 1547–1558 ( 1996).

    Article  CAS  Google Scholar 

  23. Straight, A. F., Marshall, W. F., Sedat, J. W. & Murray, A. W. Mitosis in living budding yeast: anaphase A but no metaphase plate. Science 277, 574– 578 (1997).

    Article  CAS  Google Scholar 

  24. Gorbsky, G. J., Chen, R. H. & Murray, A. W. Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase. J. Cell Biol. 141, 1193–1205 (1998).

    Article  CAS  Google Scholar 

  25. Vaisberg, E. A., Koonce, M. P. & McIntosh, J. R. Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. J. Cell Biol. 123, 849–858 (1993).

    Article  CAS  Google Scholar 

  26. Savoian, M. S., Goldberg, M. L. & Rieder, C. L. The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants. Nature Cell Biol. 2, 948–952 (2000).

    Article  CAS  Google Scholar 

  27. Orozco, J. T. et al. Movement of motor and cargo along cilia. Nature 398, 674 (1999).

    Article  CAS  Google Scholar 

  28. Signor, D. et al. Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans. J. Cell Biol. 147, 519–530 ( 1999).

    Article  CAS  Google Scholar 

  29. Skibbens, R. V., Skeen, V. P. & Salmon, E. D. Directional instability of kinetochore motility during chromosome congression and segregation in mitotic newt lung cells: a push-pull mechanism. J. Cell Biol. 122, 859–875 (1993).

    Article  CAS  Google Scholar 

  30. Vernos, I. & Karsenti, E. in Dynamics of Cell Division (ed. Glover, S. A. E. a. D. M.) 97–123 (Oxford Univ. Press, 1998).

    Google Scholar 

  31. Yen, T. J., Li, G., Schaar, B. T., Szilak, I. & Cleveland, D. W. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature 359, 536–539 (1992).

    Article  CAS  Google Scholar 

  32. Wood, K. W., Sakowicz, R., Goldstein, L. S. & Cleveland, D. W. CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. Cell 91, 357– 366 (1997).

    Article  CAS  Google Scholar 

  33. Desai, A., Verma, S., Mitchison, T. J. & Walczak, C. E. Kin I kinesins are microtubule-destabilizing enzymes. Cell 96, 69–78 (1999).

    Article  CAS  Google Scholar 

  34. Lombillo, V. A., Nislow, C., Yen, T. J., Gelfand, V. I. & McIntosh, J. R. Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro. J. Cell Biol. 128, 107–115 (1995).

    Article  CAS  Google Scholar 

  35. Maney, T., Hunter, A. W., Wagenbach, M. & Wordeman, L. Mitotic centromere-associated kinesin is important for anaphase chromosome segregation. J. Cell Biol. 142, 787– 801 (1998).

    Article  CAS  Google Scholar 

  36. Zhai, Y., Kronebusch, P. J. & Borisy, G. G. Kinetochore microtubule dynamics and the metaphase–anaphase transition. J. Cell Biol. 131, 721– 734 (1995).

    Article  CAS  Google Scholar 

  37. Nicklas, R. B. A quantitative comparison of cellular motile systems. Cell Motil. 4, 1–5 (1984 ).

    Article  CAS  Google Scholar 

  38. Foe, V. E. & Alberts, B. M. Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J. Cell Sci. 61 , 31–70 (1983).

    CAS  PubMed  Google Scholar 

  39. Signor, D., Wedaman, K. P., Rose, L. S. & Scholey, J. M. Two heteromeric kinesin complexes in chemosensory neurons and sensory cilia of Caenorhabditis elegans. Mol. Biol. Cell 10 , 345–360 (1999).

    Article  CAS  Google Scholar 

  40. Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W. & Scholey, J. M. Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos. Nature Cell Biol. 1, 51–54 (1999).

    Article  CAS  Google Scholar 

  41. Francis-Lang, H., Minden, J., Sullivan, W. & Oegema, K. Live confocal analysis with fluorescently labeled proteins. Methods Mol. Biol. 122, 223–237 (1995).

    Google Scholar 

  42. Sharp, D. J. et al. The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles . J. Cell Biol. 144, 125– 138 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. B. Vallee and C. Echeverri for human p50 dynamitin cDNA, R. Saint and W. Sullivan for GFP–histone fly stock, and T. Orr-Weaver for GFP–MeiS332 fly stock. This work was supported by NIH grant no. GM55507 to J.M.S. and NIH postdoctoral fellowship no. GM19262 to D.J.S.

Author information

Authors and Affiliations

  1. Section of Molecular and Cellular Biology, University of California-Davis, 1 Shields Avenue, Davis, 95616, California, USA

    David J. Sharp, Gregory C. Rogers & Jonathan M. Scholey

Authors
  1. David J. Sharp
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gregory C. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jonathan M. Scholey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jonathan M. Scholey.

Supplementary information

Movie 1

Control GFP–histone. Time-lapse movie showing chromatid-to-pole movements during anaphase on an individual mitotic spindle from a control-injected embryo. Chromosomes are labelled with GFP–histone (green); spindle microtubules are labelled with rhodamine–tubulin (red). Images were acquired at intervals of ~8.4 s. (MOV 171 kb)

Movie 2

p50 GFP–histone. Time-lapse movie showing chromatid-to-pole movements (equivalent time points to those shown in Movie 1) on an individual spindle from an embryo in which dynein has been inhibited by micro-injection of p50-dynamitin. Chromosomes are labelled with GFP–histone (green); spindle microtubules are labelled with rhodamine–tubulin (red). Note that sister chromatids do not separate but instead decondense at the metaphase plate. (MOV 376 kb)

Movie 3

Control MeiS332. Time-lapse movie (images were acquired at 8.4-s intervals) showing kinetochore-to-pole movements on a single mitotic spindle from a control-injected embryo. Kinetochores are labelled with GFP–MeiS332 (green); spindle microtubules are labelled with rhodamine–tubulin (red). The poleward movement of an individual kinetochore is tracked by an arrow. (MOV 137 kb)

Movie 4

p50 MeiS332. Time-lapse movie showing kinetochore-to-pole movements following the inhibition of dynein by micro-injection of p50 dynamitin. Kinetochores are labelled with GFP–MeiS332 (green); spindle microtubules are labelled with rhodamine–tubulin (red). The movement of an individual kinetochore is tracked by an arrow. Note that under these conditions the kinetochores fail to move polewards from the metaphase plate. (MOV 163 kb)

Movie 5

p50-injection gradient. Time-lapse movie showing the entire surface of a p50-injected embryo and illustrating the gradient of phenotypes observed after injection of dynein inhibitors. The p50-injection site is marked in the first several frames; chromosomes are shown in green and microtubules are shown in red. Note that on nuclei near to the injection site, centrosomes do not completely separate and prophase arrest occurs. Further from the injection site, however, bipolar spindles form but fail to separate their chromosomes. (MOV 5007 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sharp, D., Rogers, G. & Scholey, J. Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos. Nat Cell Biol 2, 922–930 (2000). https://doi.org/10.1038/35046574

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35046574

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing