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.

  • Letter
  • Published:

aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division

Nature Cell Biology volume 13, pages 1132–1138 (2011)Cite this article

Subjects

Abstract

The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically1,2,3. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3–PAR-6–aPKC polarity complex1,2,3,4,5,6. However, molecular interactions between polarity proteins and LIN-5–GPR–Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: PKC-3 aPKC directs LIN-5 phosphorylation in vivo.
Figure 2: PKC-3 phosphorylates LIN-5 at the anterior cortex of the one-cell embryo.
Figure 3: PKC-3 phosphorylates LIN-5 in mitosis and requires CDK-1.
Figure 4: PKC-3-mediated phosphorylation of LIN-5 directs mitotic spindle positioning in the one-cell embryo.
Figure 5: Model illustrating how phosphorylation of LIN-5 by PKC-3 may control the position of the mitotic spindle in the one-cell embryo.

Similar content being viewed by others

References

  1. Knoblich, J. A. Asymmetric cell division: recent developments and their implications for tumour biology. Nat. Rev. Mol. Cell Biol. 11, 849–860 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Siller, K. H. & Doe, C. Q. Spindle orientation during asymmetric cell division. Nat. Cell Biol. 11, 365–374 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Galli, M. & van den Heuvel, S. Determination of the cleavage plane in early C. elegans embryos. Annu. Rev. Genet. 42, 389–411 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Srinivasan, D. G., Fisk, R. M., Xu, H. & van den Heuvel, S. A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C. elegans. Genes Dev. 17, 1225–1239 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Colombo, K. et al. Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos. Science 300, 1957–1961 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Gotta, M. et al. Asymmetrically distributed C. elegans homologs of AGS3/PINS control spindle position in the early embryo. Curr. Biol. 13, 1029–1037 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Etemad-Moghadam, B., Guo, S. & Kemphues, K. J. Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos. Cell 83, 743–752 (1995).

    Article  CAS  PubMed  Google Scholar 

  8. Hung, T. J. & Kemphues, K. J. PAR-6 is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in Caenorhabditis elegans embryos. Development 126, 127–135 (1999).

    CAS  PubMed  Google Scholar 

  9. Tabuse, Y. et al. Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans. Development 125, 3607–3614 (1998).

    CAS  PubMed  Google Scholar 

  10. Cuenca, A. A. et al. Polarization of the C. elegans zygote proceeds via distinct establishment and maintenance phases. Development 130, 1255–1265 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Boyd, L. et al. PAR-2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C. elegans embryos. Development 122, 3075–3084 (1996).

    CAS  PubMed  Google Scholar 

  12. Guo, S. & Kemphues, K. J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611–620 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Hao, Y., Boyd, L. & Seydoux, G. Stabilization of cell polarity by the C. elegans RING protein PAR-2. Dev. Cell 10, 199–208 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Grill, S. W., Gonczy, P., Stelzer, E. H. & Hyman, A. A. Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630–633 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. van der Voet, M. et al. NuMA-related LIN-5, ASPM-1, calmodulin and dynein promote meiotic spindle rotation independently of cortical LIN-5/GPR/Gα. Nat. Cell Biol. 11, 269–277 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Nguyen-Ngoc, T., Afshar, K. & Gonczy, P. Coupling of cortical dynein and Gα proteins mediates spindle positioning in Caenorhabditis elegans. Nat. Cell Biol. 9, 1294–1302 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Bowman, S. K. et al. The Drosophila NuMA homolog Mud regulates spindle orientation in asymmetric cell division. Dev. Cell 10, 731–742 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Du, Q. & Macara, I. G. Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119, 503–516 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Izumi, Y. et al. Drosophila Pins-binding protein Mud regulates spindle-polarity coupling and centrosome organization. Nat. Cell Biol. 8, 586–593 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Siller, K. H., Cabernard, C. & Doe, C. Q. The NuMA-related Mud protein binds Pins and regulates spindle orientation in Drosophila neuroblasts. Nat. Cell Biol. 8, 594–600 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Krijgsveld, J. et al. Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat. Biotechnol. 21, 927–931 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Goulding, M. B. et al. Control of nuclear centration in the C. elegans zygote by receptor-independent Gα signaling and myosin II. J. Cell Biol. 178, 1177–1191 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Labbe, J. C., McCarthy, E. K. & Goldstein, B. The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly. J. Cell Biol. 167, 245–256 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wirtz-Peitz, F., Nishimura, T. & Knoblich, J. A. Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization. Cell 135, 161–173 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Frokjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lorson, M. A., Horvitz, H. R. & van den Heuvel, S. LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans. J. Cell Biol. 148, 73–86 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Park, D. H. & Rose, L. S. Dynamic localization of LIN-5 and GPR-1/2 to cortical force generation domains during spindle positioning. Dev. Biol. 315, 42–54 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Panbianco, C. et al. A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning. Dev. Cell 15, 198–208 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rose, L. S. & Kemphues, K. The let-99 gene is required for proper spindleorientation during cleavage of the C. elegans embryo. Development 125, 1337–1346 (1998).

    CAS  PubMed  Google Scholar 

  30. Krueger, L. E., Wu, J. C., Tsou, M. F. & Rose, L. S. LET-99 inhibits lateral posterior pulling forces during asymmetric spindle elongation in C. elegans embryos. J. Cell Biol. 189, 481–495 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hao, Y. et al. Par3 controls epithelial spindle orientation by aPKC-mediated phosphorylation of apical Pins. Curr. Biol. 20, 1809–1818 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Boxem, M. et al. A protein domain-based interactome network for C. elegans early embryogenesis. Cell 134, 534–545 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Vidal, M. et al. Reverse two-hybrid and one-hybrid systems to detect dissociation of protein–protein and DNA-protein interactions. Proc. Natl Acad. Sci. USA 93, 10315–10320 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Fisk Green, R. et al. Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5. Mol. Genet. Genom. 271, 532–544 (2004).

    Article  CAS  Google Scholar 

  35. Golden, A. et al. Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans. J. Cell Biol. 151, 1469–1482 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Anderson, D. C., Gill, J. S., Cinalli, R. M. & Nance, J. Polarization of the C. elegans embryo by RhoGAP-mediated exclusion of PAR-6 from cell contacts. Science 320, 1771–1774 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Pinkse, M. W. et al. Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster. J. Proteome Res. 7, 687–697 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  39. Olsen, J. V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Gouw, J. W. et al. In vivo stable isotope labeling of fruit flies reveals post-transcriptional regulation in the maternal-to-zygotic transition. Mol. Cell Proteom. 8, 1566–1578 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Krijgsveld for help in the initial phases of the project, and A. Thomas and M. Tanenbaum for critically reading the manuscript, helpful advice and support throughout the project. We thank members of the van den Heuvel and Boxem laboratories for fruitful discussions. We are grateful to C. Frokjaer-Jensen and E. Jorgensen for reagents and advice on MosSCI. M.G. was supported by a predoctoral fellowship from the Boehringer Ingelheim Fonds and foundation ‘De Drie Lichten’ in the Netherlands. This work was also supported by the Netherlands Proteomics Centre, which is part of the Netherlands Genomics Initiative.

Author information

Authors and Affiliations

  1. Developmental Biology, Utrecht University, 3584 CH Utrecht, The Netherlands

    Matilde Galli, Vincent Portegijs, Mike Boxem & Sander van den Heuvel

  2. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CH Utrecht, The Netherlands

    Javier Muñoz & Albert J. R. Heck

  3. Netherlands Proteomics Center, The Netherlands

    Javier Muñoz & Albert J. R. Heck

  4. Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany

    Stephan W. Grill

  5. Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany

    Stephan W. Grill

Authors
  1. Matilde Galli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Javier Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vincent Portegijs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mike Boxem
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stephan W. Grill
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Albert J. R. Heck
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sander van den Heuvel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.G. and S.v.d.H. designed the project and wrote the manuscript. J.M. and A.J.R.H. helped design the quantitative mass spectrometry experiments, J.M. carried out the mass spectrometry experiments and analysed the data. M.G. carried out all other experiments. V.P. and M.B. provided help in molecular cloning and yeast two-hybrid experiments. S.W.G. guided the spindle severing experiments. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Sander van den Heuvel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2466 kb)

Supplementary Table 1

Supplementary Information (XLS 63 kb)

Supplementary Table 2

Supplementary Information (XLS 657 kb)

Supplementary Table 3

Supplementary Information (XLS 489 kb)

Supplementary Table 4

Supplementary Information (PDF 203 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Galli, M., Muñoz, J., Portegijs, V. et al. aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division. Nat Cell Biol 13, 1132–1138 (2011). https://doi.org/10.1038/ncb2315

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2315

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