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In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics

Nature Cell Biology volume 15, pages 853–859 (2013)Cite this article

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Abstract

Cytokinesis in many eukaryotes involves the contraction of an actomyosin-based contractile ring1,2. However, the detailed mechanism of contractile ring contraction is not fully understood. Here, we establish an experimental system to study contraction of the ring to completion in vitro. We show that the contractile ring of permeabilized fission yeast cells undergoes rapid contraction in an ATP- and myosin-II-dependent manner in the absence of other cytoplasmic constituents. Surprisingly, neither actin polymerization nor its disassembly is required for contraction of the contractile ring, although addition of exogenous actin-crosslinking proteins blocks ring contraction. Using contractile rings generated from fission yeast cytokinesis mutants, we show that not all proteins required for assembly of the ring are required for its contraction in vitro. Our work provides the beginnings of the definition of a minimal contraction-competent cytokinetic ring apparatus.

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Figure 1: Contractile rings in cell ghosts.
Figure 2: ATP stimulates rapid ring contraction in vitro.
Figure 3: Myosin-II drives ring contraction in vitro.
Figure 4: Contribution of actin dynamics to ring contraction in vitro.
Figure 5: Effect of tropomyosin and actin-crosslinking proteins on ring contraction in vitro.

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References

  1. Glotzer, M. The molecular requirements for cytokinesis. Science 307, 1735–1739 (2005).

    Article  CAS  Google Scholar 

  2. Pollard, T. D. Mechanics of cytokinesis in eukaryotes. Curr. Opin. Cell Biol. 22, 50–56 (2010).

    Article  CAS  Google Scholar 

  3. Mabuchi, I. Biochemical aspects of cytokinesis. Int. Rev. Cytol. 101, 175–213 (1986).

    Article  CAS  Google Scholar 

  4. Schroeder, T. E. in Molecules and Cell Movements (eds Inoué, S. & Stephens, R. E.) 305–334 (Raven, 1975).

    Google Scholar 

  5. Mabuchi, I., Tsukita, S., Tsukita, S. & Sawai, T. Cleavage furrow isolated from newt eggs: contraction, organization of the actin filaments, and protein components of the furrow. Proc. Natl Acad. Sci. USA 85, 5966–5970 (1988).

    Article  CAS  Google Scholar 

  6. Robinson, D. N. & Spudich, J. A. Mechanics and regulation of cytokinesis. Curr. Opin. Cell Biol. 16, 182–188 (2004).

    Article  CAS  Google Scholar 

  7. Pollard, T. D. & Wu, J. Q. Understanding cytokinesis: lessons from fission yeast. Nat. Rev. Mol. Cell Biol. 11, 149–155 (2010).

    Article  CAS  Google Scholar 

  8. Wolfe, B. A. & Gould, K. L. Split decisions: Coordinating cytokinesis in yeast. Trends Cell Biol. 15, 10–18 (2005).

    Article  CAS  Google Scholar 

  9. Mishra, M. et al. Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast. J. Cell Sci. 125, 3850–3857 (2012).

    Article  CAS  Google Scholar 

  10. Young, B. A., Buser, C. & Drubin, D. G. Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus. Cytoskeleton 67, 13–22 (2010).

    CAS  PubMed  Google Scholar 

  11. Motegi, F., Arai, R. & Mabuchi, I. Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell. Mol. Biol. Cell 12, 1367–1380 (2001).

    Article  CAS  Google Scholar 

  12. Pelham, R. J. & Chang, F. Actin dynamics in the contractile ring during cytokinesis in fission yeast. Nature 419, 82–86 (2002).

    Article  CAS  Google Scholar 

  13. Wu, J. Q., Kuhn, J. R., Kovar, D. R. & Pollard, T. D. Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev. Cell 5, 723–734 (2003).

    Article  CAS  Google Scholar 

  14. Arai, R. & Mabuchi, I. F-actin ring formation and the role of F-actin cables in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 115, 887–898 (2002).

    CAS  PubMed  Google Scholar 

  15. Kamasaki, T., Osumi, M. & Mabuchi, I. Three-dimensional arrangement of F-actin in the contractile ring of fission yeast. J. Cell Biol. 178, 765–771 (2007).

    Article  CAS  Google Scholar 

  16. Mabuchi, I. & Okuno, M. The effect of myosin antibody on the division of starfish blastomeres. J. Cell Biol. 74, 251–263 (1977).

    Article  CAS  Google Scholar 

  17. Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299, 1743–1747 (2003).

    Article  CAS  Google Scholar 

  18. Nakamura, M., Kakuda, T., Oba, Y., Ojika, M. & Nakamura, H. Synthesis of biotinylated xestoquinone that retains inhibitory activity against Ca2+ ATPase of skeletal muscle myosin. Bioorg. Med. Chem. 11, 3077–3082 (2003).

    Article  CAS  Google Scholar 

  19. Kitayama, C., Sugimoto, A. & Yamamoto, M. Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe. J. Cell Biol. 137, 1309–1319 (1997).

    Article  CAS  Google Scholar 

  20. Motegi, F., Nakano, K., Kitayama, C., Yamamoto, M. & Mabuchi, I. Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe. FEBS Lett. 420, 161–166 (1997).

    Article  CAS  Google Scholar 

  21. Bezanilla, M., Forsburg, S. L. & Pollard, T. D. Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis. Mol. Biol. Cell 8, 2693–2705 (1997).

    Article  CAS  Google Scholar 

  22. Balasubramanian, M. et al. Isolation and characterization of new fission yeast cytokinesis mutants. Genetics 149, 1265–1275 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lord, M. & Pollard, T. D. UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II. J. Cell Biol. 167, 315–325 (2004).

    Article  CAS  Google Scholar 

  24. Wong, K. C., Naqvi, N. I., Iino, Y., Yamamoto, M. & Balasubramanian, M. K. Fission yeast Rng3p: An UCS-domain protein that mediates myosin II assembly during cytokinesis. J. Cell Sci. 113, 2421–2432 (2000).

    CAS  PubMed  Google Scholar 

  25. McCollum, D., Balasubramanian, M. K., Pelcher, L. E., Hemmingsen, S. M. & Gould, K. L. Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis. J. Cell Biol. 130, 651–660 (1995).

    Article  CAS  Google Scholar 

  26. Mendes Pinto, I., Rubinstein, B., Kucharavy, A., Unruh, J. R. & Li, R. Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis. Dev. Cell 22, 1247–1260 (2012).

    Article  CAS  Google Scholar 

  27. Nakano, K. & Mabuchi, I. Actin-depolymerizing protein Adf1 is required for formation and maintenance of the contractile ring during cytokinesis in fission yeast. Mol. Biol. Cell 17, 1933–1945 (2006).

    Article  CAS  Google Scholar 

  28. Kovar, D. R., Kuhn, J. R., Tichy, A. L. & Pollard, T. D. The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin. J. Cell Biol. 161, 875–887 (2003).

    Article  CAS  Google Scholar 

  29. Chang, F., Drubin, D. & Nurse, P. cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J. Cell Biol. 137, 169–182 (1997).

    Article  CAS  Google Scholar 

  30. Roberts-Galbraith, R. H. et al. Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site. Mol. Cell 39, 86–99 (2010).

    Article  CAS  Google Scholar 

  31. Balasubramanian, M. K., Helfman, D. M. & Hemmingsen, S. M. A new tropomyosin essential for cytokinesis in the fission yeast S. pombe. Nature 360, 84–87 (1992).

    Article  CAS  Google Scholar 

  32. Stark, B. C., Sladewski, T. E., Pollard, L. W. & Lord, M. Tropomyosin and myosin-II cellular levels promote actomyosin ring assembly in fission yeast. Mol. Biol. Cell 21, 989–1000 (2010).

    Article  CAS  Google Scholar 

  33. Eng, K., Naqvi, N. I., Wong, K. C. & Balasubramanian, M. K. Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body. Curr. Biol. 8, 611–621 (1998).

    Article  CAS  Google Scholar 

  34. Nakano, K., Satoh, K., Morimatsu, A., Ohnuma, M. & Mabuchi, I. Interactions among a fimbrin, a capping protein, and an actin-depolymerizing factor in organization of the fission yeast actin cytoskeleton. Mol. Biol. Cell 12, 3515–3526 (2001).

    Article  CAS  Google Scholar 

  35. Takaine, M., Numata, O. & Nakano, K. Fission yeast IQGAP arranges actin filaments into the cytokinetic contractile ring. EMBO J. 28, 3117–3131 (2009).

    Article  CAS  Google Scholar 

  36. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

    Article  CAS  Google Scholar 

  37. Bähler, J. et al. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).

    Article  Google Scholar 

  38. Kobori, H., Yamada, N., Taki, A. & Osumi, M. Actin is associated with the formation of the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 94, 635–646 (1989).

    PubMed  Google Scholar 

  39. Kamasaki, T., Arai, R., Osumi, M. & Mabuchi, I. Directionality of F-actin cables changes during the fission yeast cell cycle. Nat. Cell Biol. 7, 916–917 (2005).

    Article  CAS  Google Scholar 

  40. Arai, R., Nakano, K. & Mabuchi, I. Subcellular localization and possible function of actin, tropomyosin and actin-related protein 3 (Arp3) in the fission yeast Schizosaccharomyces pombe. Eur. J. Cell Biol. 76, 288–295 (1998).

    Article  CAS  Google Scholar 

  41. Fiske, C. H. & Subbarow, Y. The Colorimetric determination of phosphorus. J. Biol. Chem. 66, 375–400 (1925).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank D. McCollum (University of Massachusetts Medical School, USA), K. Gould (Vanderbilt University, USA), J-Q. Wu (Ohio State University, USA), V. Simanis (ISREC, Switzerland), J. Bähler (University College London, UK), T. D. Pollard (Yale University, USA), T. Toda (Cancer Research, UK), I. Hagan (Paterson Institute, UK), F. Chang (Columbia University, USA), D. Mulvihill (University of Kent, UK), P. Perez (CSIC, Spain), Y. Hiraoka (Osaka University, Japan), K. Nakano (University of Tsukuba, Japan), S. Oliferenko (TLL, Singapore), and M. Sato (Waseda University, Japan) and the Yeast Genetic Resource Center Japan for providing plasmids and strains, Y. Oba and M. Ojika (Nagoya University, Japan) for their kind gift of DXQ, Y. Toyoshima (University of Tokyo, Japan) for her kind gift of kinesin, R. Amikura for help in electron microscopy, and S. Oliferenko, S. Bulchand and D. Subramanian for critical reading of this manuscript. We thank K. Gull (Oxford University, UK) for antibodies. We thank M. Sevugan for technical assistance. This work was supported by a Japan Society for Promotion of Science (JSPS) grant-in-aid for scientific research (I.M., #22247031); JSPS research fellowships for young scientists (J.K.); NUS JSPS collaborative grant (I.M. and M.B.), Temasek Life Sciences Laboratory and Singapore Millennium Foundation (M.B. and M.M.); visiting scientist fellowship from the Gakushuin University (M.M.) and Mechanobiology Institute (M.B., R.S. and Y.H.).

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  1. Mithilesh Mishra and Jun Kashiwazaki: These authors contributed equally to this work

Authors and Affiliations

  1. Temasek Life Sciences Laboratory, The National University of Singapore, 1 Research Link, Singapore 117604, Singapore

    Mithilesh Mishra & Mohan K. Balasubramanian

  2. Department of Life Sciences, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan

    Jun Kashiwazaki, Tomoko Takagi & Issei Mabuchi

  3. Mechanobiology Institute, The National University of Singapore, 117543, Singapore

    Ramanujam Srinivasan, Yinyi Huang & Mohan K. Balasubramanian

  4. Department of Biological Sciences, The National University of Singapore, 117411, Singapore

    Mohan K. Balasubramanian

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Contributions

I.M. (in vitro activation and biochemistry) and M.K.B. (establishment of ring assembly in protoplasts and mutant studies) conceived the study, I.M., M.M., M.K.B. and J.K. designed the experiments. I.M., J.K., M.M., T.T., R.S. and Y.H. conducted the experiments. J.K., I.M. and M.M. analysed the data. M.K.B., M.M., J.K. and I.M. wrote the manuscript.

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Correspondence to Mohan K. Balasubramanian or Issei Mabuchi.

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The authors declare no competing financial interests.

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ATP drives ring contraction in vitro.

A contracting ring in the presence of 0.5 mM ATP at 25 °C corresponds to the frames shown in Fig. 2a. X-Y and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 199 kb)

ATP drives ring contraction.

A contracting ring in the presence of 0.01 mM ATP at 25 °C corresponds to the frames shown in Fig. 2c. X–Y and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 118 kb)

AMP-PNP does not induce ring contraction in vitro.

Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 2d. XY and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 1038 kb)

The plasma membrane of Rlc1-3xGFP expressing cell ghosts stained with FM4-64 during contraction in vitro.

Experiment was done at 25 °C. The membrane did not ingress as the ring contracts. The video corresponds to the frames shown in Fig. 2g. (MOV 450 kb)

Incomplete actomyosin arcs do not show ATP-dependent contraction in vitro.

The GFP fluorescence decays the presence of 0.5 mM ATP but the actomyosin arc do not show any contraction at 25 °C. The video corresponds to the frames shown in Fig. 2j. X–Y and Y–Z images were shown as non-tilted and 90°-tilted maximum projections of Z-stacks, respectively. (MOV 329 kb)

Contractile ring of myo2-E1ts undergoes slow in vitro contraction even at the permissive temperature of 25 °C.

The video corresponds to the frames shown in Fig. 3b. (MOV 105 kb)

Contractile ring of myp2 null mutant (myp2Δ) in vitro.

Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 3b. (MOV 287 kb)

Contractile ring contraction of the double mutant of myo2 and myp2 (myo2-E1ts myp2Δ) in vitro.

These rings did not show any contraction and instead fragmented and disassembled in presence of ATP at 25 °C. The video corresponds to the frames shown in Fig. 3b. (MOV 912 kb)

Addition of Jasplakinolide does not affect ring contraction in vitro.

Experiment was done at 25 °C. (MOV 548 kb)

ADF/cofilin (Adf1p) is not required for ring contraction in vitro.

Cell ghosts from adf1-1ts were held at 36 °C for 15 min before ATP addition. (MOV 534 kb)

LatA has minimal effect on ring contraction in vitro.

Experiment was done at 25 °C. (MOV 515 kb)

Formin (Cdc12p) is not required for ring contraction in vitro.

Experiment was done at 36 °C. Cell ghosts from cdc12-112ts were held at 36 °C for 15 min before ATP addition. (MOV 204 kb)

F-BAR protein (Cdc15p) is not required for ring contraction in vitro.

Experiment was done at 36 °C. Cell ghosts from cdc15-140ts were held at 36 °C for 15 minutes before ATP addition. (MOV 571 kb)

Tropomyosin (Cdc8p) is required for integrity and contraction of ring in vitro.

Cell ghosts from cdc8-110ts were held at 36 °C for 15 min before ATP addition. The video corresponds to the frames shown in Fig. 5a. (MOV 105 kb)

Addition of 1 μM purified N-terminal region of IQGAP (Rng2Ns) completely blocks ring contraction in vitro.

Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 5b. (MOV 253 kb)

Addition of 0.9 μM purified fimbrin (Fim1p) completely blocks ring contraction in vitro.

Experiment was done at 25 °C. The video corresponds to the frames shown in Fig. 5c. (MOV 212 kb)

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Mishra, M., Kashiwazaki, J., Takagi, T. et al. In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics. Nat Cell Biol 15, 853–859 (2013). https://doi.org/10.1038/ncb2781

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