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.

Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast

Nature Cell Biology volume 10, pages 395–406 (2008)Cite this article

Abstract

By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

Figure 1: Inn1 is required for cell division and associates with the contractile actomyosin ring.
Figure 2: Localization of Inn1 is dependent on components of the actomyosin ring.
Figure 3: Inn1 is not required for assembly or contraction of the actomyosin ring, or for correct localization of the septin component Cdc12.
Figure 4: Inn1 is required for division of the cytoplasm at the end of mitosis.
Figure 5: Inn1 is required for ingression of the plasma membrane during cytokinesis.
Figure 6: The Inn1 protein has a C2 domain at its N-terminus, and the rest of the protein is rich in PXXP motifs.
Figure 7: The C2 domain of Inn1 is essential for ingression of the plasma membrane during cytokinesis and the remainder of the protein is required to recruit Inn1 to the bud-neck.
Figure 8: Artificial recruitment of the C2 domain of Inn1 to the actomyosin ring is sufficient to allow ingression of the plasma membrane in cells lacking the INN1 gene.

References

  1. Albertson, R., Riggs, B. & Sullivan, W. Membrane traffic: a driving force in cytokinesis. Trends Cell Biol. 15, 92–101 (2005).

    CAS  Article  PubMed  Google Scholar 

  2. Strickland, L. I. & Burgess, D. R. Pathways for membrane trafficking during cytokinesis. Trends Cell Biol. 14, 115–118 (2004).

    CAS  Article  PubMed  Google Scholar 

  3. Burgess, D. R. & Chang, F. Site selection for the cleavage furrow at cytokinesis. Trends Cell Biol. 15, 156–162 (2005).

    CAS  Article  PubMed  Google Scholar 

  4. D'Avino, P. P., Savoian, M. S. & Glover, D. M. Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy. J. Cell Sci. 118, 1549–1558 (2005).

    CAS  Article  PubMed  Google Scholar 

  5. Tolliday, N., Bouquin, N. & Li, R. Assembly and regulation of the cytokinetic apparatus in budding yeast. Curr. Opin. Microbiol. 4, 690–695 (2001).

    CAS  Article  PubMed  Google Scholar 

  6. Balasubramanian, M. K., Bi, E. & Glotzer, M. Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells. Curr. Biol. 14, R806–R818 (2004).

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  8. Barr, F. A. & Gruneberg, U. Cytokinesis: placing and making the final cut. Cell 131, 847–860 (2007).

    CAS  Article  PubMed  Google Scholar 

  9. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).

    CAS  Article  PubMed  Google Scholar 

  10. Winzeler, E. A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    CAS  Article  PubMed  Google Scholar 

  11. Kanemaki, M., Sanchez-Diaz, A., Gambus, A. & Labib, K. Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. Nature 423, 720–725 (2003).

    CAS  Article  PubMed  Google Scholar 

  12. Dohmen, R. J., Wu, P. & Varshavsky, A. Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science 263, 1273–1276 (1994).

    CAS  Article  PubMed  Google Scholar 

  13. Labib, K., Tercero, J. A. & Diffley, J. F. X. Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000).

    CAS  Article  PubMed  Google Scholar 

  14. Cheeseman, I. M. et al. Implication of a novel multiprotein Dam1p complex in outer kinetochore function. J. Cell Biol. 155, 1137–1145 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Enquist-Newman, M. et al. Dad1p, third component of the Duo1p/Dam1p complex involved in kinetochore function and mitotic spindle integrity. Mol. Biol. Cell 12, 2601–2613 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Euskirchen, G. M. Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae. Eukaryot. Cell 1, 229–240 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Janke, C., Ortiz, J., Tanaka, T. U., Lechner, J. & Schiebel, E. Four new subunits of the Dam1–Duo1 complex reveal novel functions in sister kinetochore biorientation. EMBO J. 21, 181–193 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Li, Y. et al. The mitotic spindle is required for loading of the DASH complex onto the kinetochore. Genes Dev. 16, 183–197 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Pinsky, B. A., Tatsutani, S. Y., Collins, K. A. & Biggins, S. An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase. Dev. Cell 5, 735–745 (2003).

    CAS  Article  PubMed  Google Scholar 

  20. Scharfenberger, M. et al. Nsl1p is essential for the establishment of bipolarity and the localization of the Dam–Duo complex. EMBO J. 22, 6584–6597 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Lippincott, J. & Li, R. Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution. J. Cell Biol. 143, 1947–1960 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Vallen, E. A., Caviston, J. & Bi, E. Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae. Mol. Biol. Cell 11, 593–611 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Dobbelaere, J. & Barral, Y. Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science 305, 393–396 (2004).

    CAS  Article  PubMed  Google Scholar 

  24. Norden, C. et al. The NoCut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage. Cell 125, 85–98 (2006).

    CAS  Article  PubMed  Google Scholar 

  25. Whistler, J. L. & Rine, J. Ras2 and Ras1 protein phosphorylation in Saccharomyces cerevisiae. J. Biol. Chem. 272, 18790–18800 (1997).

    CAS  Article  PubMed  Google Scholar 

  26. Cho, W. & Stahelin, R. V. Membrane binding and subcellular targeting of C2 domains. Biochim. Biophys. Acta 1761, 838–849 (2006).

    CAS  Article  PubMed  Google Scholar 

  27. Cho, W. & Stahelin, R. V. Membrane-protein interactions in cell signaling and membrane trafficking. Annu. Rev. Biophys. Biomol. Struct. 34, 119–151 (2005).

    CAS  Article  PubMed  Google Scholar 

  28. Hurley, J. H. Membrane binding domains. Biochim. Biophys. Acta 1761, 805–811 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. VerPlank, L. & Li, R. Cell cycle-regulated trafficking of Chs2 controls actomyosin ring stability during cytokinesis. Mol. Biol. Cell 16, 2529–2543 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Zhang, G., Kashimshetty, R., Ng, K. E., Tan, H. B. & Yeong, F. M. Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to mother-daughter neck via the secretory pathway in budding yeast. J. Cell Biol. 174, 207–220 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Roh, D. H., Bowers, B., Schmidt, M. & Cabib, E. The septation apparatus, an autonomous system in budding yeast. Mol. Biol. Cell 13, 2747–2759 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Chuang, J. S. & Schekman, R. W. Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p. J. Cell Biol. 135, 597–610 (1996).

    CAS  Article  PubMed  Google Scholar 

  33. Martens, S., Kozlov, M. M. & McMahon, H. T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

    CAS  Article  PubMed  Google Scholar 

  34. Aspenstrom, P., Fransson, A. & Richnau, N. Pombe Cdc15 homology proteins: regulators of membrane dynamics and the actin cytoskeleton. Trends Biochem. Sci. 31, 670–679 (2006).

    Article  PubMed  Google Scholar 

  35. Field, C. M. & Alberts, B. M. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J. Cell Biol. 131, 165–178 (1995).

    CAS  Article  PubMed  Google Scholar 

  36. Oegema, K., Savoian, M. S., Mitchison, T. J. & Field, C. M. Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis. J. Cell Biol. 150, 539–552 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Echard, A., Hickson, G. R., Foley, E. & O'Farrell, P. H. Terminal cytokinesis events uncovered after an RNAi screen. Curr. Biol. 14, 1685–1693 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Somma, M. P., Fasulo, B., Cenci, G., Cundari, E. & Gatti, M. Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol. Biol. Cell 13, 2448–2460 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Straight, A. F., Field, C. M. & Mitchison, T. J. Anillin binds nonmuscle myosin II and regulates the contractile ring. Mol. Biol. Cell 16, 193–201 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Zhao, W. M. & Fang, G. Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis. J. Biol. Chem. 280, 33516–33524 (2005).

    CAS  Article  PubMed  Google Scholar 

  41. Losev, E. et al. Golgi maturation visualized in living yeast. Nature 441, 1002–1006 (2006).

    CAS  Article  PubMed  Google Scholar 

  42. Spurr, A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31–43 (1969).

    CAS  Article  PubMed  Google Scholar 

  43. Reynolds, E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Gambus, A. et al. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nature Cell Biol. 8, 358–366 (2006).

    CAS  Article  PubMed  Google Scholar 

  45. Rost, B., Yachdav, G. & Liu, J. The PredictProtein server. Nucleic Acids Res 32, W321–W326 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Rizo, J. & Sudhof, T. C. C2-domains, structure and function of a universal Ca2+-binding domain. J. Biol. Chem. 273, 15879–15882 (1998).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded by Cancer Research U.K., from whom K.L. received a Senior Cancer Research Fellowship, and by the EMBO Young Investigator Programme. A.S.D. received a Marie Curie personal training fellowship from the European Union. We are very grateful to S. Bagley for much advice and help with time-lapse video microscopy, B. Glick for advice regarding the use of concanavalin A, J. Wiedenmann for the eQFP construct, and E. Schiebel for plasmids and helpful comments on the manuscript. A.S.D. thanks A. Sanchez-Sanfructuoso and J. Diaz-Marcos for their support.

Author information

Author notes

  1. Richard Jones

    Present address: Current address: NextGen Sciences, Inc., 4401 Varsity Drive, Suite E, Ann Arbor, MI 48108, USA.,

  2. Gislene Pereira

    Present address: Current address: German Cancer Research Centre, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany.,

  3. Ricky Edmondson

    Present address: Current address: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 W.Markham #776, Little Rock, AR 72205, USA.,

Affiliations

  1. Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

    Alberto Sanchez-Diaz, Vanessa Marchesi, Stephen Murray, Gislene Pereira, Terry Allen & Karim Labib

  2. FDA-NCTR, 3900 NCTR Road, Jefferson, 72079, AR, USA

    Richard Jones & Ricky Edmondson

Authors
  1. Alberto Sanchez-Diaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vanessa Marchesi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stephen Murray
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richard Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gislene Pereira
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ricky Edmondson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Terry Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Karim Labib
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V. M. generated the strains that are described in Supplementary Information, Fig. S1a and b, and screened these strains for defects in cell cycle progression to generate the corrsponding data in Supplementary Information, Fig. 1c. S. M. performed the electron microscopy that is summarized in Fig. 4a. R. J. was responsible for the mass spectrometry data in Fig. 1e. G. P. made the initial observations of the localization of Inn–GFP and taught A. S. D. how to prepare samples for fluorescence microscopy. K. L. made the strains shown in Fig. 8a, b, c. A. S. D. performed all other experiments.

Corresponding author

Correspondence to Karim Labib.

Supplementary information

Supplementary Information

Supplementary Figures S1, S2, S3, S4 and S5 (PDF 2225 kb)

Supplementary Information

Supplementary Movie 1 (MOV 1307 kb)

Supplementary Information

Supplementary Movie 2 (MOV 282 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sanchez-Diaz, A., Marchesi, V., Murray, S. et al. Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast. Nat Cell Biol 10, 395–406 (2008). https://doi.org/10.1038/ncb1701

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1701

Further reading

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