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• W2710677853 abstract "Centrioles are microtubule-based cylinders essential for the formation of centrosomes and cilia. A recent study provides a new cell-free assay that reconstitutes the initial structure formed during centriole assembly — the cartwheel — and proposes a new model for its formation and growth. Centrioles are microtubule-based cylinders essential for the formation of centrosomes and cilia. A recent study provides a new cell-free assay that reconstitutes the initial structure formed during centriole assembly — the cartwheel — and proposes a new model for its formation and growth. As Richard Feynman said, “What I cannot create, I do not understand”. This quote is particularly pertinent to scientists studying the assembly of cellular organelles. While much work has been carried out in whole cells with the aim of understanding the molecular mechanisms governing the formation of whole organelles, their in vitro reconstruction has lagged behind. This is particularly true for a one billion-year-old tiny cellular structure, the centriole, which is essential for the generation of two organelles, cilia and centrosomes. Cilia and centrosomes are involved in many critical cellular processes, such as cell motility and cell division. Abnormalities in their structure and in their number cause multiple diseases, including cancer, microcephaly and ciliopathies. In a recent study in Nature Communications [1Guichard P. Hamel V. Le Guennec M. Banterle N. Iacovache I. Nemcikova V. Fluckiger I. Goldie K.N. Stahlberg H. Levy D. et al.Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.Nat. Commun. 2017; 8: 14813Crossref PubMed Scopus (56) Google Scholar], a team led by Pierre Gönczy presents a new cell-free assay that has enabled them to reliably produce in vitro the initial structure formed during centriole assembly — the cartwheel. The feasibility of assembling this structure in vitro, in a reproducible manner, provides several avenues for the study of centriole assembly. In this paper, the authors have already revealed novel molecular and engineering principles of this process. Centrioles are 250 nm wide and ∼500 nm long microtubule-based cylinders exhibiting a nine-fold symmetry and a proximo-distal polarity, with the presence of a cartwheel and appendages at the proximal and distal ends, respectively (Figure 1A). Recent cryo-electron tomography analysis of the basal body of the symbiotic flagellate Trichonympha [2Guichard P. Desfosses A. Maheshwari A. Hachet V. Dietrich C. Brune A. Ishikawa T. Sachse C. Gonczy P. Cartwheel architecture of Trichonympha basal body.Science. 2012; 337: 553Crossref PubMed Scopus (72) Google Scholar, 3Guichard P. Hachet V. Majubu N. Neves A. Demurtas D. Olieric N. Fluckiger I. Yamada A. Kihara K. Nishida Y. et al.Native architecture of the centriole proximal region reveals features underlying its 9-fold radial symmetry.Curr. Biol. 2013; 23: 1620-1628Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar] unravelled the structural organisation of the cartwheel, which is typically composed of stacked rings with a periodicity of ∼8.5 nm in the central region and ∼17 nm in the outer region (Figure 1A). Each ring comprises a 22 nm diameter central hub from which nine radial spokes emanate to contact the microtubules via the pinhead structure (Figure 1A). Due to its nine-fold symmetry and its presence at an initial stage of centriole assembly, the cartwheel is thought to govern the beginning of centriole biogenesis and to help in establishing the nine-fold symmetry. The SAS-6 protein is the major molecular component of the cartwheel and has an essential and evolutionarily conserved role in cartwheel formation [4van Breugel M. Hirono M. Andreeva A. Yanagisawa H.A. Yamaguchi S. Nakazawa Y. Morgner N. Petrovich M. Ebong I.O. Robinson C.V. et al.Structures of SAS-6 suggest its organization in centrioles.Science. 2011; 331: 1196-1199Crossref PubMed Scopus (234) Google Scholar, 5Kitagawa D. Vakonakis I. Olieric N. Hilbert M. Keller D. Olieric V. Bortfeld M. Erat M.C. Fluckiger I. Gonczy P. et al.Structural basis of the 9-fold symmetry of centrioles.Cell. 2011; 144: 364-375Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 6Rodrigues-Martins A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. DSAS-6 organizes a tube-like centriole precursor, and its absence suggests modularity in centriole assembly.Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 7Jerka-Dziadosz M. Gogendeau D. Klotz C. Cohen J. Beisson J. Koll F. Basal body duplication in Paramecium: the key role of Bld10 in assembly and stability of the cartwheel.Cytoskeleton. 2010; 67: 161-171Google Scholar, 8Nakazawa Y. Hiraki M. Kamiya R. Hirono M. SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole.Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar]. Recent studies built a structural model of the cartwheel in which the nine-fold symmetrical rings are composed of nine amino-terminal dimers of SAS-6 forming the hub, from which nine carboxy-terminal coiled-coil SAS-6 dimers radiate to constitute the spokes [4van Breugel M. Hirono M. Andreeva A. Yanagisawa H.A. Yamaguchi S. Nakazawa Y. Morgner N. Petrovich M. Ebong I.O. Robinson C.V. et al.Structures of SAS-6 suggest its organization in centrioles.Science. 2011; 331: 1196-1199Crossref PubMed Scopus (234) Google Scholar, 5Kitagawa D. Vakonakis I. Olieric N. Hilbert M. Keller D. Olieric V. Bortfeld M. Erat M.C. Fluckiger I. Gonczy P. et al.Structural basis of the 9-fold symmetry of centrioles.Cell. 2011; 144: 364-375Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar]. Nevertheless, SAS-6 is unlikely to be the sole driver of the formation and stacking of cartwheel rings since SAS-6 alone can only form hubs in vitro and cannot account for the total spoke–pinhead length. Indeed, other players have been shown to be part of the cartwheel structure and to interact with each other, in particular Bld10/CEP135, Ana2/STIL and CPAP [9Lin Y.C. Chang C.W. Hsu W.B. Tang C.J. Lin Y.N. Chou E.J. Wu C.T. Tang T.K. Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly.EMBO J. 2013; 32: 1141-1154Crossref PubMed Scopus (143) Google Scholar, 10Stevens N.R. Roque H. Raff J.W. DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement.Dev. Cell. 2010; 19: 913-919Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 11Hiraki M. Nakazawa Y. Kamiya R. Hirono M. Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole.Curr. Biol. 2007; 17: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Qiao R. Cabral G. Lettman M.M. Dammermann A. Dong G. SAS-6 coiled-coil structure and interaction with SAS-5 suggest a regulatory mechanism in C. elegans centriole assembly.EMBO J. 2012; 31: 4334-4347Crossref PubMed Scopus (45) Google Scholar, 13Tang C.J. Lin S.Y. Hsu W.B. Lin Y.N. Wu C.T. Lin Y.C. Chang C.W. Wu K.S. Tang T.K. The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.EMBO J. 2011; 30: 4790-4804Crossref PubMed Scopus (182) Google Scholar]. While the structural and molecular composition of the cartwheel is well characterised, the minimal components needed to form the structure and the mechanisms governing ring stacking are not known. In this recent paper, Guichard et al. [1Guichard P. Hamel V. Le Guennec M. Banterle N. Iacovache I. Nemcikova V. Fluckiger I. Goldie K.N. Stahlberg H. Levy D. et al.Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.Nat. Commun. 2017; 8: 14813Crossref PubMed Scopus (56) Google Scholar] explore the mechanisms governing the assembly and stacking of cartwheel rings in Chlamydomonas reinhardtii using an in vitro reconstruction of the core cartwheel (i.e. hub and spokes). The authors first set out to identify the native structural organisation of the C. reinhardtii cartwheel because, until now, the architectural features of the cartwheel were mostly only known from Trichonympha basal bodies [2Guichard P. Desfosses A. Maheshwari A. Hachet V. Dietrich C. Brune A. Ishikawa T. Sachse C. Gonczy P. Cartwheel architecture of Trichonympha basal body.Science. 2012; 337: 553Crossref PubMed Scopus (72) Google Scholar, 3Guichard P. Hachet V. Majubu N. Neves A. Demurtas D. Olieric N. Fluckiger I. Yamada A. Kihara K. Nishida Y. et al.Native architecture of the centriole proximal region reveals features underlying its 9-fold radial symmetry.Curr. Biol. 2013; 23: 1620-1628Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar]. Here, using cryo-electron tomography on purified centrioles, the authors demonstrated that the C. reinhardtii cartwheel exhibits architectural features (hub, spokes, recruitment of A-microtubules) akin to those of the Trichonympha cartwheel, as well as two previously uncharacterized densities — D1 and D2 — along the length of the spokes (Figure 1B). With these results, the authors identified the different cartwheel features to be reconstituted in vitro and demonstrated that the architecture of the core cartwheel is likely conserved throughout evolution. To study cartwheel formation, the authors developed cell-free assays that involve analysing, by cryo-EM, assemblies that form upon dialysis of soluble recombinant proteins. Confirming previous studies in C. reinhardtii and Danio rerio [4van Breugel M. Hirono M. Andreeva A. Yanagisawa H.A. Yamaguchi S. Nakazawa Y. Morgner N. Petrovich M. Ebong I.O. Robinson C.V. et al.Structures of SAS-6 suggest its organization in centrioles.Science. 2011; 331: 1196-1199Crossref PubMed Scopus (234) Google Scholar, 5Kitagawa D. Vakonakis I. Olieric N. Hilbert M. Keller D. Olieric V. Bortfeld M. Erat M.C. Fluckiger I. Gonczy P. et al.Structural basis of the 9-fold symmetry of centrioles.Cell. 2011; 144: 364-375Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar], the authors showed that recombinant SAS-6 self-assembles into ring structures similar to the central hub of the cartwheel but fails to form the core cartwheel structure because spokes are not well-organised and the peripheral densities (D1 and D2) are not detectable (Figure 1C). This result reinforced the idea that interacting partners of CrSAS-6 are required for cartwheel formation. Guichard et al. [1Guichard P. Hamel V. Le Guennec M. Banterle N. Iacovache I. Nemcikova V. Fluckiger I. Goldie K.N. Stahlberg H. Levy D. et al.Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.Nat. Commun. 2017; 8: 14813Crossref PubMed Scopus (56) Google Scholar] further hypothesized that Bld10p could be the missing piece for several reasons: Bld10p is required for cartwheel formation in C. reinhardtii [11Hiraki M. Nakazawa Y. Kamiya R. Hirono M. Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole.Curr. Biol. 2007; 17: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar]; the authors showed that Bl10p localises to the cartwheel by structured illumination microscopy; and Cep135/Bld10 interacts with SAS-6 in human cells through its carboxy-terminal domain [9Lin Y.C. Chang C.W. Hsu W.B. Tang C.J. Lin Y.N. Chou E.J. Wu C.T. Tang T.K. Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly.EMBO J. 2013; 32: 1141-1154Crossref PubMed Scopus (143) Google Scholar]. After incubating full-length CrSAS-6 together with the carboxyl terminus of Bld10p, the authors observed the formation of 3D core cartwheel-like structures displaying the native features of the cartwheel (central hub of 22 nm and detection of the peripheral densities D1 and D2; Figure 1C). Altogether, these results demonstrated that CrSAS-6 alone cannot self-assemble into a core cartwheel structure but instead requires, in C. reinhardtii, the interaction with Bld10p to achieve this process. The authors hypothesized that spoke organisation, the presence of peripheral densities (D1 and D2), and ring stacking could either require Bld10p presence or be inhibited by a domain of CrSAS-6 that is masked upon interaction with Bld10p. Further experiments conducted by the authors supported the second hypothesis, as they observed that recombinant SAS-6 lacking its carboxy-terminal domain can form 3D lattices composed of core cartwheel-like structures, albeit lacking D2 and often showing eight- instead of nine-fold symmetry. With this study, Guichard et al. [1Guichard P. Hamel V. Le Guennec M. Banterle N. Iacovache I. Nemcikova V. Fluckiger I. Goldie K.N. Stahlberg H. Levy D. et al.Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.Nat. Commun. 2017; 8: 14813Crossref PubMed Scopus (56) Google Scholar] demonstrated for the first time that SAS-6 itself has the ability to generate stacked entities in solution akin to the in vivo cartwheels, but that its carboxy-terminal domain negatively regulates this property. This is of particular interest as it suggests a regulatory mechanism by which the number of cartwheels produced is restricted, even in the presence of excess SAS-6. Sub-tomogram averaging and cryo-tomogram analysis of assemblies formed by CrSAS-6 lacking its carboxy-terminal domain or by full-length CrSAS-6 upon interaction with the Bld10p carboxyl terminus further confirmed that the 3D cartwheels formed in vitro have the same vertical organisation as the in vivo cartwheels of Trichonympha (∼8 nm and ∼15–17 nm central and peripheral periodicity, respectively, and fusion of spokes at the periphery; Figure 1C). With this cell-free assay, the authors successfully reconstituted 3D cartwheel structures with native architectural features that enabled them to further investigate the mechanisms of cartwheel ring stacking. Analysis of cartwheel height distribution revealed an average height of 107 nm, similar to the height observed in vivo in Chlamydomonas [14O'Toole E.T. Dutcher S.K. Site-specific basal body duplication in Chlamydomonas.Cytoskeleton. 2014; 71: 108-118Crossref Scopus (35) Google Scholar]. Interestingly, peaks in height frequencies were observed every 8 nm or 17 nm, therefore suggesting that the building blocks of the cartwheel are either single or double SAS-6 rings. The authors further developed a mathematical model to fit their experimental data that subsequently allowed them to propose a new model of cartwheel assembly characterised by a polarised incorporation of pairs of SAS-6 rings onto a single seed ring (Figure 2). With this paper, Guichard et al. [1Guichard P. Hamel V. Le Guennec M. Banterle N. Iacovache I. Nemcikova V. Fluckiger I. Goldie K.N. Stahlberg H. Levy D. et al.Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.Nat. Commun. 2017; 8: 14813Crossref PubMed Scopus (56) Google Scholar] provide a new assay that reconstitutes the core cartwheel structure in vitro. Further improvement of the assay, with better spatiotemporal resolution, will further elucidate the mechanisms governing ring assembly and stacking as well as those controlling cartwheel height. Moreover, this will help to answer critical questions regarding centriole evolution and why centriole assembly is limited in space and number, i.e. close to existing centrioles and to one per existing centriole. Spatial restriction of cartwheel assembly at the centrosomes might in part result from differences in the oligomerisation state of SAS-6 between the cytosolic and the centrosomal fractions. In human cells, cytosolic SAS-6 exists mostly in homodimeric form, whereas in the centrosome, where SAS-6 seems to be a hundred times more concentrated than in the cytoplasm, this protein forms higher-order structures [15Keller D. Orpinell M. Olivier N. Wachsmuth M. Mahen R. Wyss R. Hachet V. Ellenberg J. Manley S. Gonczy P. Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells.J. Cell Biol. 2014; 204: 697-712Crossref PubMed Scopus (58) Google Scholar]. Guichard et al. [1Guichard P. Hamel V. Le Guennec M. Banterle N. Iacovache I. Nemcikova V. Fluckiger I. Goldie K.N. Stahlberg H. Levy D. et al.Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.Nat. Commun. 2017; 8: 14813Crossref PubMed Scopus (56) Google Scholar] propose that the presence of Bld10 at the centrosome masks the SAS-6 carboxy-terminal domain, therefore catalysing cartwheel assembly there. To further understand ring stacking, it is ultimately necessary to study the kinetics of the process. In practical terms, this could involve looking at ring assembly and stacking rates under different SAS-6 and/or Bld10 concentrations. Coupled with mechanistic models, this should provide insights into the principles governing cartwheel assembly, for example, whether there is cooperativity or whether stack height has an effect on growth, thereby regulating centriole height and number. Concurrently, shifting from probabilistic to more mechanistic models should help explain the principles underlying the assembly process. Understanding these mechanisms will facilitate further dissection in vivo — for example, allowing the investigation of whether cartwheel height has a biological role regarding microtubule nucleation and centriole elongation. Finally, this assay provides a foundation for building the whole centriole structure. The door is now open to identifying the minimal set of proteins required to form the full cartwheel (hub, spokes and pinhead) and to anchor microtubules." @default.
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• W2710677853 title "Centrosome Assembly: Reconstructing the Core Cartwheel Structure In Vitro" @default.
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