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Studying Centriole Duplication and Elongation in Human Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2101))

Abstract

Centrioles assemble centrosomes and cilia/flagella, which are microtubule-based structures with key roles in cell division, polarity, motility, and signaling. Centriole biogenesis is a tightly regulated process, and deregulation of centriole numbers and structure can have dramatic consequences for cellular function and integrity. However, their small size poses a challenge to study them. Here, we describe protocols that allow the identification and assessment of true centrioles and that provide straightforward strategies to study the role of new candidate proteins in centriole duplication and elongation.

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References

  1. Bornens M (2002) Centrosome composition and microtubule anchoring mechanisms. Curr Opin Cell Biol 14(1):25–34

    CAS  PubMed  Google Scholar 

  2. Fu J, Lipinszki Z, Rangone H, Min M, Mykura C, Chao-Chu J, Schneider S, Dzhindzhev NS, Gottardo M, Riparbelli MG, Callaini G, Glover DM (2016) Conserved molecular interactions in centriole-to-centrosome conversion. Nat Cell Biol 18(1):87–99

    CAS  PubMed  Google Scholar 

  3. Piperno G, LeDizet M, Chang XJ (1987) Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol 104(2):289–302

    CAS  PubMed  Google Scholar 

  4. Gundersen GG, Bulinski JC (1986) Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis. J Cell Biol 102(3):1118–1126

    CAS  PubMed  Google Scholar 

  5. Eddé B, Rossier J, Le Caer JP, Desbruyères E, Gros F, Denoulet P (1990) Posttranslational glutamylation of alpha-tubulin. Science 247(4938):83–85

    PubMed  Google Scholar 

  6. Bobinnec Y, Khodjakov A, Mir LM, Rieder CL, Eddé B, Bornens M (1998) Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells. J Cell Biol 143(6):1575–1589

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Bobinnec Y, Moudjou M, Fouquet JP, Desbruyères E, Eddé B, Bornens M (1998) Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells. Cell Motil Cytoskeleton 39(3):223–232

    CAS  PubMed  Google Scholar 

  8. Bettencourt-Dias M, Hildebrandt F, Pellman D, Woods G, Godinho SA (2011) Centrosomes and cilia in human disease. Trends Genet 27(8):307–315

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Tanos BE, Yang HJ, Soni R, Wang WJ, Macaluso FP, Asara JM, Tsou MF (2013) Centriole distal appendages promote membrane docking, leading to cilia initiation. Genes Dev 27(2):163–168

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Piel M, Meyer P, Khodjakov A, Rieder CL, Bornens M (2000) The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J Cell Biol 149(2):317–330

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Nigg EA, Holland AJ (2018) Once and only once: mechanisms of centriole duplication and their deregulation in disease. Nat Rev Mol Cell Biol 19(5):297–312

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Sonnen KF, Gabryjonczyk AM, Anselm E, Stierhof YD, Nigg EA (2013) Human Cep192 and Cep152 cooperate in Plk4 recruitment and centriole duplication. J Cell Sci 126(Pt 14):3223–3233

    CAS  PubMed  Google Scholar 

  13. Cizmecioglu O, Arnold M, Bahtz R, Settele F, Ehret L, Haselmann-Weiss U, Antony C, Hoffmann I (2010) Cep152 acts as a scaffold for recruitment of Plk4 and CPAP to the centrosome. J Cell Biol 191(4):731–739

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hatch EM, Kulukian A, Holland AJ, Cleveland DW, Stearns T (2010) Cep152 interacts with Plk4 and is required for centriole duplication. J Cell Biol 191(4):721–729

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Dzhindzhev NS, Tzolovsky G, Lipinszki Z, Schneider S, Lattao R, Fu J, Debski J, Dadlez M, Glover DM (2014) Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation. Curr Biol 24(21):2526–2532

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Arquint C, Gabryjonczyk AM, Imseng S, Böhm R, Sauer E, Hiller S, Nigg EA, Maier T (2015) STIL binding to Polo-box 3 of PLK4 regulates centriole duplication. Elife 4. https://doi.org/10.7554/eLife.07888

  17. Ohta M, Ashikawa T, Nozaki Y, Kozuka-Hata H, Goto H, Inagaki M, Oyama M, Kitagawa D (2014) Direct interaction of Plk4 with STIL ensures formation of a single procentriole per parental centriole. Nat Commun 5:5267

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Moyer TC, Clutario KM, Lambrus BG, Daggubati V, Holland AJ (2015) Binding of STIL to Plk4 activates kinase activity to promote centriole assembly. J Cell Biol 209(6):863–878

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Ohta M, Watanabe K, Ashikawa T, Nozaki Y, Yoshiba S, Kimura A, Kitagawa D (2018) Bimodal binding of STIL to Plk4 controls proper centriole copy number. Cell Rep 23(11):3160–3169.e4

    CAS  PubMed  Google Scholar 

  20. Arquint C, Nigg EA (2016) The PLK4-STIL-SAS-6 module at the core of centriole duplication. Biochem Soc Trans 44(5):1253–1263

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Azimzadeh J, Marshall WF (2010) Building the centriole. Curr Biol 20(18):R816–R825

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Vorobjev IA, Chentsov YS (1982) Centrioles in the cell cycle. I. Epithelial cells. J Cell Biol 93(3):938–949

    CAS  PubMed  Google Scholar 

  23. Kong D, Farmer V, Shukla A, James J, Gruskin R, Kiriyama S, Loncarek J (2014) Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles. J Cell Biol 206(7):855–865

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Uzbekov R, Alieva I (2018) Who are you, subdistal appendages of centriole? Open Biol 8(7):180062

    PubMed  PubMed Central  Google Scholar 

  25. Marthiens V, Rujano MA, Pennetier C, Tessier S, Paul-Gilloteaux P, Basto R (2013) Centrosome amplification causes microcephaly. Nat Cell Biol 15(7):731–740

    CAS  PubMed  Google Scholar 

  26. Levine MS, Bakker B, Boeckx B, Moyett J, Lu J, Vitre B, Spierings DC, Lansdorp PM, Cleveland DW, Lambrechts D, Foijer F, Holland AJ (2017) Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals. Dev Cell 40(3):313–322.e5

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Godinho SA, Pellman D (2014) Causes and consequences of centrosome abnormalities in cancer. Philos Trans R Soc Lond Ser B Biol Sci 369(1650):20130467

    Google Scholar 

  28. Leidel S, Delattre M, Cerutti L, Baumer K, Gönczy P (2005) SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells. Nat Cell Biol 7(2):115–125

    CAS  PubMed  Google Scholar 

  29. Comartin D, Gupta GD, Fussner E, Coyaud É, Hasegan M, Archinti M, Cheung SW, Pinchev D, Lawo S, Raught B, Bazett-Jones DP, Lüders J, Pelletier L (2013) CEP120 and SPICE1 cooperate with CPAP in centriole elongation. Curr Biol 23(14):1360–1366

    CAS  PubMed  Google Scholar 

  30. Kleylein-Sohn J, Westendorf J, Le Clech M, Habedanck R, Stierhof YD, Nigg EA (2007) Plk4-induced centriole biogenesis in human cells. Dev Cell 13(2):190–120

    CAS  PubMed  Google Scholar 

  31. Habedanck R, Stierhof YD, Wilkinson CJ, Nigg EA (2005) The Polo kinase Plk4 functions in centriole duplication. Nat Cell Biol 7(11):1140–1146

    CAS  PubMed  Google Scholar 

  32. Kimura M, Yoshioka T, Saio M, Banno Y, Nagaoka H, Okano Y (2013) Mitotic catastrophe and cell death induced by depletion of centrosomal proteins. Cell Death Dis 4(4):e603

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang G, Jiang Q, Zhang C (2014) The role of mitotic kinases in coupling the centrosome cycle with the assembly of the mitotic spindle. J Cell Sci 127(Pt 19):4111–4122

    CAS  PubMed  Google Scholar 

  34. Bettencourt-Dias M, Rodrigues-Martins A, Carpenter L, Riparbelli M, Lehmann L, Gatt MK, Carmo N, Balloux F, Callaini G, Glover DM (2005) SAK/PLK4 is required for centriole duplication and flagella development. Curr Biol 15(24):2199–2207

    CAS  PubMed  Google Scholar 

  35. Holland AJ, Fachinetti D, Zhu Q, Bauer M, Verma IM, Nigg EA, Cleveland DW (2012) The autoregulated instability of Polo-like kinase 4 limits centrosome duplication to once per cell cycle. Genes Dev 26(24):2684–2689

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Coelho PA, Bury L, Shahbazi MN, Liakath-Ali K, Tate PH, Wormald S, Hindley CJ, Huch M, Archer J, Skarnes WC, Zernicka-Goetz M, Glover DM (2015) Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse. Open Biol 5(12):150209

    PubMed  PubMed Central  Google Scholar 

  37. Balczon R, Bao L, Zimmer WE, Brown K, Zinkowski RP, Brinkley BR (1995) Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells. J Cell Biol 130(1):105–115

    CAS  PubMed  Google Scholar 

  38. Kuriyama R, Terada Y, Lee KS, Wang CL (2007) Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2. J Cell Sci 120(Pt 14):2444–2453

    CAS  PubMed  Google Scholar 

  39. Loncarek J, Hergert P, Magidson V, Khodjakov A (2008) Control of daughter centriole formation by the pericentriolar material. Nat Cell Biol 10(3):322–328

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Tang CJ, Fu RH, Wu KS, Hsu WB, Tang TK (2009) CPAP is a cell-cycle regulated protein that controls centriole length. Nat Cell Biol 11(7):825–831

    CAS  PubMed  Google Scholar 

  41. Schmidt TI, Kleylein-Sohn J, Westendorf J, Le Clech M, Lavoie SB, Stierhof YD, Nigg EA (2009) Control of centriole length by CPAP and CP110. Curr Biol 19(12):1005–1011

    CAS  PubMed  Google Scholar 

  42. Lin YN, Wu CT, Lin YC, Hsu WB, Tang CJ, Chang CW, Tang TK (2013) CEP120 interacts with CPAP and positively regulates centriole elongation. J Cell Biol 202(2):211–219

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  44. Pihan GA (2013) Centrosome dysfunction contributes to chromosome instability, chromoanagenesis, and genome reprograming in cancer. Front Oncol 3:277

    PubMed  PubMed Central  Google Scholar 

  45. Lopes CAM, Mesquita M, Cunha AI, Cardoso J, Carapeta S, Laranjeira C, Pinto AE, Pereira-Leal JB, Dias-Pereira A, Bettencourt-Dias M, Chaves P (2018) Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis. J Cell Biol 217(7):2353–2363

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Marteil G, Guerrero A, Vieira AF, de Almeida BP, Machado P, Mendonça S, Mesquita M, Villarreal B, Fonseca I, Francia ME, Dores K, Martins NP, Jana SC, Tranfield EM, Barbosa-Morais NL, Paredes J, Pellman D, Godinho SA, Bettencourt-Dias M (2018) Over-elongation of centrioles in cancer promotes centriole amplification and chromosome missegregation. Nat Commun 9(1):1258

    PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We are grateful to E. Nigg for cell lines. C.A.M. Lopes and C. Peneda are funded by Fundação para a Ciência e a Tecnologia (SFRH/BPD/124127/2016 and PD/BD/128004/2016, respectively). The M. Bettencourt-Dias laboratory is funded by Fundação Calouste Gulbenkian and the European Research Council (ERC-2015-CoG-683258).

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Peneda, C., Lopes, C.A.M., Bettencourt-Dias, M. (2020). Studying Centriole Duplication and Elongation in Human Cells. In: Maiato, H. (eds) Cytoskeleton Dynamics. Methods in Molecular Biology, vol 2101. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0219-5_10

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  • DOI: https://doi.org/10.1007/978-1-0716-0219-5_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0218-8

  • Online ISBN: 978-1-0716-0219-5

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