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Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material

Abstract

The centrosome is the main microtubule organization centre of animal cells. It is composed of a centriole pair surrounded by pericentriolar material (PCM). Traditionally described as amorphous, the architecture of the PCM is not known, although its intricate mode of assembly alludes to the presence of a functional, hierarchical structure. Here we used subdiffraction imaging to reveal organizational features of the PCM. Interphase PCM components adopt a concentric toroidal distribution of discrete diameter around centrioles. Positional mapping of multiple non-overlapping epitopes revealed that pericentrin (PCNT) is an elongated molecule extending away from the centriole. We find that PCM components occupy separable spatial domains within mitotic PCM that are maintained in the absence of microtubule nucleation complexes and further implicate PCNT and CDK5RAP2 in the organization and assembly of PCM. Globally, this work highlights the role of higher-order PCM organization in the regulation of centrosome assembly and function.

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Figure 1: The interphase centrosome exhibits a layered organization.
Figure 2: Positional mapping of PCM components.
Figure 3: The interphase toroid structure is similar in size in G1 and G2.
Figure 4: PCM spatial domains in mitotic centrosomes.
Figure 5: PCM spatial domains in fragmented mitotic centrosomes.
Figure 6: PCNT is required for PCM association with centrioles during mitosis.
Figure 7: Lattice formation occurs in interphase cells.
Figure 8: Organizational features of the interphase centrosome.

References

  1. Lampert, F. & Westermann, S. A blueprint for kinetochore—new insights into the molecular mechanics of cell division. Nat. Rev. Mol. Cell Biol. 12, 407–412 (2011).

    CAS  Article  Google Scholar 

  2. Cheeseman, I. M. & Desai, A. Molecular architecture of the kinetochore-microtubule interface. Nat. Rev. 9, 33–46 (2008).

    CAS  Article  Google Scholar 

  3. Bettencourt-Dias, M. & Glover, D. M. Centrosome biogenesis and function: centrosomics brings new understanding. Nat. Rev. 8, 451–463 (2007).

    CAS  Article  Google Scholar 

  4. Wan, X. et al. Protein architecture of the human kinetochore microtubule attachment site. Cell 137, 672–684 (2009).

    CAS  Article  Google Scholar 

  5. Nigg, E. A. & Raff, J. W. Centrioles, centrosomes, and cilia in health and disease. Cell 139, 663–678 (2009).

    CAS  Article  Google Scholar 

  6. Bettencourt-Dias, M., Hildebrandt, F., Pellman, D., Woods, G. & Godinho, S.A. Centrosomes and cilia in human disease. Trends Genet. 27, 307–315 (2011).

    CAS  Article  Google Scholar 

  7. Nigg, E. A. Centrosome aberrations: cause or consequence of cancer progression? Nat. Rev. Cancer 2, 815–825 (2002).

    CAS  Article  Google Scholar 

  8. Kleylein-Sohn, J. et al. Plk4-induced centriole biogenesis in human cells. Dev. cell 13, 190–202 (2007).

    CAS  Article  Google Scholar 

  9. Pelletier, L., O’Toole, E., Schwager, A., Hyman, A. A. & Muller-Reichert, T. Centriole assembly in Caenorhabditis elegans. Nature 444, 619–623 (2006).

    CAS  Article  Google Scholar 

  10. Ou, Y. & Rattner, J. B. The centrosome in higher organisms: structure, composition, and duplication. Int. Rev. Cytol. 238, 119–182 (2004).

    CAS  Article  Google Scholar 

  11. Dictenberg, J. B. et al. Pericentrin and γ-tubulin form a protein complex andare organized into a novel lattice at the centrosome. J. Cell Biol. 141, 163–174 (1998).

    CAS  Article  Google Scholar 

  12. Palazzo, R. E., Vogel, J. M., Schnackenberg, B. J., Hull, D. R. & Wu, X. Centrosome maturation. Curr. Top. Dev. Biol. 49, 449–470 (2000).

    CAS  Article  Google Scholar 

  13. Barr, F. A., Sillje, H. H. & Nigg, E. A. Polo-like kinases and the orchestration of cell division. Nat. Rev. Mol. Cell Biol. 5, 429–440 (2004).

    CAS  Article  Google Scholar 

  14. Petronczki, M., Lenart, P. & Peters, J. M. Polo on the rise-from mitotic entry to cytokinesis with Plk1. Dev. Cell 14, 646–659 (2008).

    CAS  Article  Google Scholar 

  15. Glover, D. M., Leibowitz, M. H., McLean, D. A. & Parry, H. Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81, 95–105 (1995).

    CAS  Article  Google Scholar 

  16. Glover, D. M., Hagan, I. M. & Tavares, A. A. Polo-like kinases: a team that plays throughout mitosis. Gen. Dev. 12, 3777–3787 (1998).

    CAS  Article  Google Scholar 

  17. Hannak, E., Kirkham, M., Hyman, A. A. & Oegema, K. Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans. J. Cell Biol. 155, 1109–1116 (2001).

    CAS  Article  Google Scholar 

  18. Moritz, M., Braunfeld, M. B., Sedat, J. W., Alberts, B. & Agard, D. A. Microtubule nucleation by γ-tubulin-containing rings in the centrosome. Nature 378, 638–640 (1995).

    CAS  Article  Google Scholar 

  19. Zheng, Y., Wong, M. L., Alberts, B. & Mitchison, T. Nucleation of microtubule assembly by a γ-tubulin-containing ring complex. Nature 378, 578–583 (1995).

    CAS  Article  Google Scholar 

  20. Khodjakov, A. & Rieder, C. L. The sudden recruitment of γ-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. J. Cell Biol. 146, 585–596 (1999).

    CAS  Article  Google Scholar 

  21. Zhu, F. et al. The mammalian SPD-2 ortholog Cep192 regulates centrosome biogenesis. Curr. Biol. 18, 136–141 (2008).

    CAS  Article  Google Scholar 

  22. Luders, J., Patel, U. K. & Stearns, T. GCP-WD is a γ-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation. Nat. Cell Biol. 8, 137–147 (2006).

    Article  Google Scholar 

  23. Haren, L. et al. NEDD1-dependent recruitment of the γ-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly. J. Cell Biol. 172, 505–515 (2006).

    CAS  Article  Google Scholar 

  24. Graser, S., Stierhof, Y. D. & Nigg, E. A. Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion. J. Cell Sci. 120, 4321–4331 (2007).

    CAS  Article  Google Scholar 

  25. Gomez-Ferreria, M. A. et al. Human Cep192 is required for mitotic centrosome and spindle assembly. Curr. Biol. 17, 1960–1966 (2007).

    CAS  Article  Google Scholar 

  26. Doxsey, S. J., Stein, P., Evans, L., Calarco, P. D. & Kirschner, M. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell 76, 639–650 (1994).

    CAS  Article  Google Scholar 

  27. Barrera, J. A. et al. CDK5RAP2 regulates centriole engagement and cohesion in mice. Dev. Cell 18, 913–926 (2010).

    CAS  Article  Google Scholar 

  28. Haren, L., Stearns, T. & Luders, J. Plk1-dependent recruitment of γ-tubulin complexes to mitotic centrosomes involves multiple PCM components. PLoS One 4, e5976 (2009).

    Article  Google Scholar 

  29. Santamaria, A. et al. Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis. Mol. Biol. Cell 18, 4024–4036 (2007).

    CAS  Article  Google Scholar 

  30. Lee, K. & Rhee, K. PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis. J. Cell Biol. 195, 1093–1101 (2011).

    CAS  Article  Google Scholar 

  31. Loncarek, J., Hergert, P., Magidson, V. & Khodjakov, A. Control of daughter centriole formation by the pericentriolar material. Nat. Cell Biol. 10, 322–328 (2008).

    CAS  Article  Google Scholar 

  32. Megraw, T. L., Kao, L. R. & Kaufman, T. C. Zygotic development without functional mitotic centrosomes. Curr. Biol. 11, 116–120 (2001).

    CAS  Article  Google Scholar 

  33. Megraw, T. L., Li, K., Kao, L. R. & Kaufman, T. C. The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila. Development 126, 2829–2839 (1999).

    CAS  PubMed  Google Scholar 

  34. Fong, K. W., Choi, Y. K., Rattner, J. B. & Qi, R. Z. CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the γ-tubulin ring complex. Mol. Biol. cell 19, 115–125 (2008).

    CAS  Article  Google Scholar 

  35. O’Connell, K. F., Maxwell, K. N. & White, J. G. The spd-2 gene is required for polarization of the anteroposterior axis and formation of the sperm asters in the Caenorhabditis elegans zygote. Dev. Biol. 222, 55–70 (2000).

    Article  Google Scholar 

  36. Pelletier, L. et al. The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication. Curr. Biol. 14, 863–873 (2004).

    CAS  Article  Google Scholar 

  37. Dix, C. I. & Raff, J. W. Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication. Curr. Biol. 17, 1759–1764 (2007).

    CAS  Article  Google Scholar 

  38. Conduit, P. T. et al. Centrioles regulate centrosome size by controlling the rate of CNN incorporation into the PCM. Curr. Biol. 20, 2178–2186 (2010).

    CAS  Article  Google Scholar 

  39. Gopalakrishnan, J. et al. Sas-4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome. Nat. Commun. 2, 359 (2011).

    Article  Google Scholar 

  40. Dobbie, I. M. et al. OMX: a new platform for multimodal, multichannel wide-field imaging. Cold Spring Harb. Protoc. 899–909 (2011).

  41. Gustafsson, M. G. et al. Three-dimensional resolution doubling in wide-fieldfluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).

    CAS  Article  Google Scholar 

  42. Schermelleh, L. et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).

    CAS  Article  Google Scholar 

  43. Gillingham, A. K. & Munro, S. The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin. EMBO Rep. 1, 524–529 (2000).

    CAS  Article  Google Scholar 

  44. Aldaz, H., Rice, L. M., Stearns, T. & Agard, D. A. Insights into microtubulenucleation from the crystal structure of human γ-tubulin. Nature 435, 523–527 (2005).

    CAS  Article  Google Scholar 

  45. Hutchins, J. R. et al. Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science 328, 593–599 (2010).

    CAS  Article  Google Scholar 

  46. Hubner, N. C. et al. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754 (2010).

    CAS  Article  Google Scholar 

  47. Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    CAS  Article  Google Scholar 

  48. Bucciarelli, E. et al. Drosophila Dgt6 interacts with Ndc80, Msps/XMAP215, and γ-tubulin to promote kinetochore-driven MT formation. Curr. Biol. 19, 1839–1845 (2009).

    CAS  Article  Google Scholar 

  49. Goshima, G., Mayer, M., Zhang, N., Stuurman, N. & Vale, R. D. Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle. J. Cell Biol. 181, 421–429 (2008).

    CAS  Article  Google Scholar 

  50. Lawo, S. et al. HAUS, the 8-subunit human Augmin complex, regulates centrosome and spindle integrity. Curr. Biol. 19, 816–826 (2009).

    CAS  Article  Google Scholar 

  51. Meireles, A. M., Fisher, K. H., Colombie, N., Wakefield, J. G. & Ohkura, H. Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis. J. Cell Biol. 184, 777–784 (2009).

    CAS  Article  Google Scholar 

  52. Uehara, R. et al. The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells. Proc. Natl Acad. Sci. USA 106, 6998–7003 (2009).

    CAS  Article  Google Scholar 

  53. Sir, J.H. et al. A primary microcephaly protein complex forms a ring around parental centrioles. Nat. Gen. 43, 1147–1153 (2011).

    CAS  Article  Google Scholar 

  54. Wang, Z. et al. Conserved motif of CDK5RAP2 mediates its localization to centrosomes and the Golgi complex. J. Biol. Chem. 285, 22658–22665 (2010).

    CAS  Article  Google Scholar 

  55. Zimmerman, W. C., Sillibourne, J., Rosa, J. & Doxsey, S. J. Mitosis-specific anchoring of γ tubulin complexes by pericentrin controls spindle organization and mitotic entry. Mol. Biol. Cell 15, 3642–3657 (2004).

    CAS  Article  Google Scholar 

  56. Mahjoub, M. R., Xie, Z. & Stearns, T. Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly. J. Cell Biol. 191, 331–346 (2010).

    CAS  Article  Google Scholar 

  57. Gomez-Ferreria, M. et al. Novel NEDD1 phosphorylation sites regulate γ-tubulin binding and mitotic spindle assembly. J. Cell Sci. advance online publication, http://dx.doi.org/10.1242/jcs.105130 (17 May 2012).

  58. Joukov, V., De Nicolo, A., Rodriguez, A., Walter, J. C. & Livingston, D.M. Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation. Proc. Natl Acad. Sci. USA 107, 21022–21027 (2010).

    CAS  Article  Google Scholar 

  59. Kim, T. et al. Novel alternatively spliced variant form of human CDK5RAP2. Cell Cycle 10, 1010–1012 (2011).

    CAS  Article  Google Scholar 

  60. Kirkham, M., Muller-Reichert, T., Oegema, K., Grill, S. & Hyman, A.A. SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112, 575–587 (2003).

    CAS  Article  Google Scholar 

  61. Bobinnec, Y. et al. Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells. Cell Motil. Cytoskeleton 39, 223–232 (1998).

    CAS  Article  Google Scholar 

  62. Basto, R. et al. Flies without centrioles. Cell 125, 1375–1386 (2006).

    CAS  Article  Google Scholar 

  63. Szollosi, A., Ris, H., Szollosi, D. & Debec, A. A centriole-free Drosophila cell line. A high voltage EM study. Eur. J. Cell Biol. 40, 100–104 (1986).

    CAS  PubMed  Google Scholar 

  64. Mennella, V. et al. Sub-diffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization. Nat. Cell Biol. 14, http://dx.doi.org/10.1038/ncb2597 (2012).

  65. Sonnen, K. F., Schermelleh, L., Leonhardt, H. & Nigg, E.A. 3D-structured illumination microscopy provides novel insight into architecture of human centrosomes. Biol. Open, advance online publication, http://dx.doi.org/10.1242/bio.20122337 (17 August 2012).

  66. Kittler, R., Heninger, A. K., Franke, K., Habermann, B. & Buchholz, F. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nat. Methods 2, 779–784 (2005).

    CAS  Article  Google Scholar 

  67. Kittler, R. et al. Genome-scale RNAi profiling of cell division in human tissue culture cells. Nat. Cell Biol. 9, 1401–1412 (2007).

    CAS  Article  Google Scholar 

  68. Kittler, R. et al. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. Proc. Natl Acad. Sci. USA 102, 2396–2401 (2005).

    CAS  Article  Google Scholar 

  69. Gustafsson, M. G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    CAS  Article  Google Scholar 

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Acknowledgements

We would like to thank members of the Pelletier laboratory for stimulating discussions during the course of this work, especially C. Yeh and J. Gonçalves for their critical reading of the manuscript, C. Holley for esiRNA production, and M. Bashkurov for help with super-resolution imaging. Furthermore, we are grateful to D. Drechsel (MPI-CBG, Dresden, Germany), S. Doxsey (University of Massachusetts Medical School, Worcester, USA), M. Gomez-Ferreria (CRG, Barcelona, Spain), J. Lüders (IRB, Barcelona, Spain), K. Rhee (Seoul National University, Korea), J. Salisbury (Mayo Clinic, Minnesota, USA) and L-H. Tsai (MIT, Cambridge, USA) for providing key reagents and Applied Precision/GE Healthcare for excellent technical support with the OMX microscope. This work was financially supported by the Canadian Cancer Society (019562), the Natural Sciences and Engineering Research Council of Canada (RGPIN-355644-2008) and a grant-in-aid from the Krembil Foundation. L.P. holds a Canada Research Chair (Tier 2) in Centrosome Biogenesis and Function. S.L. is a Vanier Canada Graduate Scholar (CIHR).

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S.L. performed the experimental work. M.H. did interphase diameter measurements and intensity line profiles. G.D.G. performed mitotic co-localization and quantitative recruitment analysis. G.D.G. and L.P. wrote the manuscript with contributions from all authors.

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Correspondence to Laurence Pelletier.

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Lawo, S., Hasegan, M., Gupta, G. et al. Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat Cell Biol 14, 1148–1158 (2012). https://doi.org/10.1038/ncb2591

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