Centriole Assembly

This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 340227

  

Centrioles are evolutionarily conserved organelles that are essential for the formation of cilia, flagella, as well as centrosomes, and which are characterized by a striking 9-fold radial symmetry of microtubules (Fig. 1). Just like the genetic material, centrioles duplicate once and only once per cell cycle. Formation of a procentriole next to each existing centriole, around a structure called the cartwheel, marks the onset of centriole duplication. Despite their importance, the mechanisms governing centriole biogenesis have remained elusive until recently.

 

Fig. 1 | Centrioles. (A) Hela Cell in mitosis. (DNA in blue, microtubules in green and centrioles marked by Centrin in red). (B) Cross section of a Trychonympha centriole revealing the characteristic 9-fold symmetry of microtubule triplets around the cartwheel.

  

We and others identified five proteins required for centriole formation in C. elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6 and SPD-2 (Gönczy, 2012). We established that centriole formation is an orderly assembly process in which these proteins are recruited in a step-wise fashion (Delattre et al.; 2006). Relatives of these proteins are crucial for centriole formation also in other organisms. Thus, we found that HsSAS-6 is necessary for procentriole formation in human cells and that excess HsSAS-6 results in supernumerary procentrioles. Therefore, regulated levels of HsSAS-6 ensure formation of a single procentriole per existing centriole during the duplication cycle (Strnad et al.; 2007).

 Fig. 2 | HsSAS-6 levels must be limited to restrict procentriole formation. U2OS cells transfected with HsSAS-6 fixed after 72 hr and stained with antibodies against centrin (green) and HsSAS-6 (red). Representative images of normal centrosome (A) or centrosome with excess centriole duplication (B).

 

In collaboration with the Steinmetz laboratory, we revealed the crystal structure and the oligomerisation properties of Chlamydomonas SAS-6 (Bld12p) by combining structural, biochemical and cell biological methods. We discovered that Bld12p can form rings in vitro that ressemble the central part of the cartwheel. This lead us to propose that oligomerization of SAS-6 proteins dictates the near-universal 9-fold symmetry of centrioles (Fig. 3) (Kitagawa et al.; 2011).

Fig. 3 | Step-wise self-assembly of SAS-6 proteins. (A) Bld12p monomer. (B) Two Bld12p proteins homodimerize through a strong interaction between the two coiled-coil domains. (C) Pairs of Bld12p homodimers undergo a weak interaction mediated by adjacent N-terminal domains. (D) Reiteration of the interaction between N-terminal domains results in the assembly of a ring of nine homodimers. (E) The Bld12p ring connects with the pinhead, thus coupling the ring with the microtubules.

The above findings left open the question whether SAS-6 rings exist in vivo. Thus, we sought to obtain a three-dimensional map of the cartwheel in the native state by using cryoelectron tomography (cryo-ET). We found that the cartwheel is a stack of central rings that exhibit a vertical periodicity of 8.5 nanometers and that is able to accommodate nine SAS-6 homodimers. Furthermore, we discovered that the spokes emanating from two such rings associate into a layer, with a vertical periodicity of 17 nanometers on the cartwheel margin (Fig. 4) (Guichard et al.; 2012).

Fig. 4 | Trichonympha cartwheel architecture. (A) Longitudinal view of the cartwheel revealing a stack of rings with a periodicity of 8.5 nm along the central hub and 17 nm along the radial spokes. (B) Close-up of the central hub ring with the fitting of Bld12p ring inside (red).

 

Our publications on this topic

P. Gönczy; G. Hatzopoulos : Centriole assembly at a glance; Journal Of Cell Science. 2019-02-01. DOI : 10.1242/jcs.228833.
B. Wolf; F. Balestra; A. Spahr; P. Gönczy : ZYG-1 promotes limited centriole amplification in the C. elegans seam lineage; DEVELOPMENTAL BIOLOGY. 2018. DOI : 10.1016/j.ydbio.2018.01.001.
P. Guichard; V. Hamel; P. Gönczy : The Rise of the Cartwheel: Seeding the Centriole Organelle; BIOESSAYS. 2018. DOI : 10.1002/bies.201700241.
N. Banterle; P. Gönczy : Centriole Biogenesis: From Identifying the Characters to Understanding the Plot; Annual Review Of Cell And Developmental Biology, Vol 33; Palo Alto: Annual Reviews, 2017. p. 23-49.
P. Guichard; V. Hamel; M. Le Guennec; N. Banterle; I. Iacovache et al. : Cell-free reconstitution reveals centriole cartwheel assembly mechanisms; Nature Communications. 2017. DOI : 10.1038/ncomms14813.
M. Graciotti; Z. Fang; K. Johnsson; P. Gönczy : Chemical Genetic Screen Identifies Natural Products that Modulate Centriole Number; ChemBioChem. 2016. DOI : 10.1002/cbic.201600327.
H. C. R. Klein; P. Guichard; V. Hamel; P. Gönczy; U. S. Schwarz : Computational support for a scaffolding mechanism of centriole assembly; Scientific Reports. 2016. DOI : 10.1038/srep27075.
J. Borrego-Pinto; K. Somogyi; M. A. Karreman; J. Koenig; T. Mueller-Reichert et al. : Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes; Journal Of Cell Biology. 2016. DOI : 10.1083/jcb.201510083.
M. Hilbert; A. Noga; D. Frey; V. Hamel; P. Guichard et al. : SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture; Nature Cell Biology. 2016. DOI : 10.1038/ncb3329.
P. Guichard; V. Hamel; A. Neves; P. Gönczy : Isolation, cryotomography, and three-dimensional reconstruction of centrioles; Centrosome & Centriole; Elsevier, 2015. p. 191-209.
K. B. Rogala; N. J. Dynes; G. N. Hatzopoulos; J. Yan; S. K. Pong et al. : The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation; eLife. 2015. DOI : 10.7554/eLife.07410.
F. R. Balestra; L. Von Tobel; P. Gönczy : Paternally contributed centrioles exhibit exceptional persistence in C-elegans embryos; Cell Research. 2015. DOI : 10.1038/cr.2015.49.
L. von Tobel; T. Mikeladze-Dvali; M. Delattre; F. R. Balestra; S. Blanchoud et al. : SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans; Plos Genetics. 2014. DOI : 10.1371/journal.pgen.1004777.
F. R. Balestra; P. Gönczy : Multiciliogenesis: multicilin directs transcriptional activation of centriole formation; Current biology : CB. 2014. DOI : 10.1016/j.cub.2014.07.006.
M. Bornens; P. Gönczy : Centrosomes back in the limelight; Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 2014. DOI : 10.1098/rstb.2013.0452.
D. Keller; M. Orpinell; N. Olivier; M. Wachsmuth; R. Mahen et al. : Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells; The Journal of cell biology. 2014. DOI : 10.1083/jcb.201307049.
P. Guichard; V. Hachet; N. Majubu; A. Neves; D. Demurtas et al. : Native Architecture of the Centriole Proximal Region Reveals Features Underlying Its 9-Fold Radial Symmetry; Current Biology. 2013. DOI : 10.1016/j.cub.2013.06.061.
F. R. Balestra; P. Strnad; I. Flückiger; P. Gönczy : Discovering Regulators of Centriole Biogenesis through siRNA-Based Functional Genomics in Human Cells; Developmental cell. 2013. DOI : 10.1016/j.devcel.2013.05.016.
P. Guichard; A. Desfosses; A. Maheshwari; V. Hachet; C. Dietrich et al. : Cartwheel Architecture of Trichonympha Basal Body; Science. 2012. DOI : 10.1126/science.1222789.
P. Gönczy : Towards a molecular architecture of centriole assembly; Nature Reviews Molecular Cell Biology. 2012. DOI : 10.1038/nrm3373.
T. Mikeladze-Dvali; L. von Tobel; P. Strnad; G. Knott; H. Leonhardt et al. : Analysis of centriole elimination during C. elegans oogenesis; Development. 2012. DOI : 10.1242/dev.075440.
D. Kitagawa; G. Kohlmaier; D. Keller; P. Strnad; F. R. Balestra et al. : Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL; Journal Of Cell Science. 2011. DOI : 10.1242/jcs.089888.
D. Kitagawa; I. Vakonakis; N. Olieric; M. Hilbert; D. Keller et al. : Structural Basis of the 9-Fold Symmetry of Centrioles; Cell. 2011. DOI : 10.1016/j.cell.2011.01.008.
D. Kitagawa; I. Flueckiger; J. Polanowska; D. Keller; J. Reboul et al. : PP2A Phosphatase Acts upon SAS-5 to Ensure Centriole Formation in C. elegans Embryos; Developmental Cell. 2011. DOI : 10.1016/j.devcel.2011.02.005.
A. Puklowski; Y. Homsi; D. Keller; M. May; S. Chauhan et al. : The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication; Nature Cell Biology. 2011. DOI : 10.1038/ncb2282.
D. Kitagawa; C. Busso; I. Fluckiger; P. Gönczy : Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos; Developmental cell. 2009. DOI : 10.1016/j.devcel.2009.11.002.
G. Kohlmaier; J. Loncarek; X. Meng; B. F. McEwen; M. M. Mogensen et al. : Overly long centrioles and defective cell division upon excess of the SAS-4-related protein CPAP; Current biology. 2009. DOI : 10.1016/j.cub.2009.05.018.
P. Strnad; S. Leidel; T. Vinogradova; U. Euteneuer; A. Khodjakov et al. : Regulated HsSAS-6 levels ensure formation of a single procentriole per centriole during the centrosome duplication cycle; Dev Cell. 2007. DOI : 10.1016/j.devcel.2007.07.004.
M. Delattre; C. Canard; P. Gönczy : Sequential Protein Recruitment in C. elegans Centriole Formation; Current Biology. 2006. DOI : 10.1016/j.cub.2006.07.059.
S. Leidel; M. Delattre; L. Cerutti; K. Baumer; P. Gönczy : SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells; Nat Cell Biol. 2005. DOI : 10.1038/ncb1220.
M. Delattre; S. Leidel; K. Wani; K. Baumer; J. Bamat et al. : Centriolar SAS-5 is required for centrosome duplication in C. elegans; Nat Cell Biol. 2004. DOI : 10.1038/ncb1146.
S. Leidel; P. Gönczy : SAS-4 is essential for centrosome duplication in C elegans and is recruited to daughter centrioles once per cell cycle; Dev Cell. 2003. DOI : 10.1016/S1534-5807(03)00062-5.