Self-assembling SAS-6 Multimer Is a Core Centriole Building Block (2010)

Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a “cartwheel” with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with ∼6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles. Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a “cartwheel” with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with ∼6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles. IntroductionCentrioles are conserved microtubule-based organelles with 9-fold symmetry organized around a central tubule and spoke structure termed the “cartwheel.” This conserved 9-fold symmetry is also shared with basal body and cilia (1.Azimzadeh J. Bornens M. Nigg E.A. Centrosomes in Development and Disease. Wiley-VCH, Weinheim, Germany2004: 93-116Google Scholar, 2.Dippell R.V. Proc. Natl. Acad. Sci. U.S.A. 1968; 61: 461-468Crossref PubMed Scopus (209) Google Scholar, 3.Vorobjev I.A. Chentsov Yu. S. J. Cell Biol. 1982; 93: 938-949Crossref PubMed Scopus (275) Google Scholar). Such architectural similarity is indicative of the role of the centriole as a template for cilia formation. Cilia are microtubule-rich cellular organelles involved in signaling pathways and sensory functions in eukaryotic cells (4.Rosenbaum J. Curr. Biol. 2002; 12: R125Abstract Full Text Full Text PDF PubMed Google Scholar, 5.Scholey J.M. Anderson K.V. Cell. 2006; 125: 439-442Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Centrioles also recruit a pericentriolar matrix to form centrosomes, major microtubule organizing centers required for cell proliferation and differentiation (6.Badano J.L. Teslovich T.M. Katsanis N. Nat. Rev. Genet. 2005; 6: 194-205Crossref PubMed Scopus (199) Google Scholar, 7.González C. Tavosanis G. Mollinari C. J. Cell Sci. 1998; 111: 2697-2706Crossref PubMed Google Scholar). In ciliated cells, one of the centrioles inherited by the daughter cell migrates to the plasma membrane after cell division to serve as a template for the cell cilium. However, when a centriole duplicates, the new centriole does not use the existing centriole as a template (8.Phillips D.M. J. Cell Biol. 1967; 33: 73-92Crossref PubMed Scopus (44) Google Scholar, 9.Rodrigues-Martins A. Riparbelli M. Callaini G. Glover D.M. Bettencourt-Dias M. Science. 2007; 316: 1046-1050Crossref PubMed Scopus (196) Google Scholar), raising the question of how the new centriole establishes its 9-fold symmetry.Centriole biogenesis is a multistep process that includes an intermediate known as the procentriole (10.Azimzadeh J. Bornens M. J. Cell Sci. 2007; 120: 2139-2142Crossref PubMed Scopus (168) Google Scholar). The procentriole has an internal cartwheel structure that is required for the stability of the centriole and the 9-fold symmetry of cilia (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The cartwheel has a central tubule from which nine spokes emanate, each attaching to centriolar microtubules (supplemental Fig. S1A). Based on the structure of the cartwheel, it is expected that the central tubule would itself display 9-fold symmetry (12.Strnad P. Gönczy P. Trends Cell Biol. 2008; 18: 389-396Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) (supplemental Fig. S1A). It has been proposed that the central tubule generates the 9-fold symmetry of the centriole (13.Cavalier-Smith T. J. Cell Sci. 1974; 16: 529-556Crossref PubMed Google Scholar, 14.Satir P. Satir B. J. Theor. Biol. 1964; 7: 123-128Crossref PubMed Scopus (11) Google Scholar). However, the composition of the central tubule and the mechanisms underlying its biogenesis remain unknown.SAS-6 is a coiled-coil protein that is required for centriole biogenesis (15.Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 16.Leidel S. Delattre M. Cerutti L. Baumer K. Gönczy P. Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (293) Google Scholar). Overexpression of SAS-6 produces multiple procentrioles (17.Strnad P. Leidel S. Vinogradova T. Euteneuer U. Khodjakov A. Gönczy P. Dev. Cell. 2007; 13: 203-213Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). In Chlamydomonas and Tetrahymena, SAS-6 localizes to the center of the cartwheel (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 18.Kilburn C.L. Pearson C.G. Romijn E.P. Meehl J.B. Giddings Jr., T.H. Culver B.P. Yates 3rd, J.R. Winey M. J. Cell Biol. 2007; 178: 905-912Crossref PubMed Scopus (132) Google Scholar). Null mutants of SAS-6 in Chlamydomonas (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and Drosophila (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 19.Rodrigues-Martins A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) lack the cartwheel. The SAS-6 null mutants of Chlamydomonas and Drosophila are also defective in establishing the 9-fold symmetry of centrioles. In Caenorhabditis elegans, RNAi studies show that SAS-6 is required for the formation of a centriole central tube. Direct evidence that SAS-6 is required for central tube assembly in worms comes from time resolved electron tomography of C. elegans embryos demonstrating that centriole formation begins with assembly of a central tube, a process that requires SAS-6 (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar). This study also implied that the tube could itself organize 9-fold symmetry, but it was not clear if the process was driven by steric constraints or was patterned by intrinsic cues on the outer surface of the tube. It was proposed that SAS-6 brings together nine pre-centriolar units called enatosomes, which then form a tube-like centriole precursor (22.Rodrigues-Martins A. Riparbelli M. Callaini G. Glover D.M. Bettencourt-Dias M. Cell Cycle. 2008; 7: 11-16Crossref PubMed Scopus (54) Google Scholar) and that SAS-6 is the repeating subunit of the central tubule (12.Strnad P. Gönczy P. Trends Cell Biol. 2008; 18: 389-396Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar).To investigate the mechanism of SAS-6 function, we employed a Drosophila model that allows for biochemical isolation and electron microscopy (EM) 2The abbreviations used are: EMelectron microscopyPCMpericentriolar materialγ-TuRCγ-tubulin ring complexγ-TuSCγ-tubulin small complexesGFPgreen fluorescent proteinPipes1,4-piperazinediethanesulfonic acidPBSphosphate-buffered saline. imaging of centrioles and cartwheels. In vertebrates the cartwheel is transient and is restricted to the “procentriole” stage (23.Alvey P.L. Cell Biol. Int. Rep. 1986; 10: 589-598Crossref PubMed Scopus (20) Google Scholar). In worms the cartwheel is not apparent (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar), and in protozoa it is restricted to the proximal end of basal bodies (18.Kilburn C.L. Pearson C.G. Romijn E.P. Meehl J.B. Giddings Jr., T.H. Culver B.P. Yates 3rd, J.R. Winey M. J. Cell Biol. 2007; 178: 905-912Crossref PubMed Scopus (132) Google Scholar, 24.Hiraki M. Nakazawa Y. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Drosophila centrioles are structurally similarly to the procentrioles of higher organisms (7.González C. Tavosanis G. Mollinari C. J. Cell Sci. 1998; 111: 2697-2706Crossref PubMed Google Scholar), and cartwheels are constitutive components of Drosophila centrioles. EM studies of in situ centrioles in Drosophila embryos reported that the cartwheel extends through the full length of the centriole (25.Callaini G. Whitfield W.G. Riparbelli M.G. Exp. Cell Res. 1997; 234: 183-190Crossref PubMed Scopus (66) Google Scholar).Here, we report that in Drosophila, the central tubule of the cartwheel has repeating subunits and that SAS-6 localizes at the central tubule. Recombinant SAS-6 produced in Escherichia coli or Pichia pastoris forms tetrameric structures that are fractionated at 7.4 S. Most of the native SAS-6 exists as 7.4 S structures, which is the most common soluble form of SAS-6. Native SAS-6 is also found as part of 50 S structures and centrosomes. Elevated levels of SAS-6 in Drosophila cells resulted in higher order structures built from multiple central tubule-like structures. Affinity purification of these high density SAS-6 complexes contained tetramers. Upon disassembly, these higher order complexes resulted in tetramers. In vitro assembly of these tetramers could then be assembled into high density complexes in the presence of embryonic extract. Collectively these data suggest that the SAS-6 tetramers serve as building blocks of a higher order central tubule at the core of centriole architecture.DISCUSSIONCentriole biogenesis includes an early intermediate known as the procentriole (10.Azimzadeh J. Bornens M. J. Cell Sci. 2007; 120: 2139-2142Crossref PubMed Scopus (168) Google Scholar). The cartwheel, a conserved centriolar scaffold, appears at the onset of procentriole assembly (31.Anderson R.G. J. Cell Biol. PubMed Scopus Google Scholar). The of a cartwheel to the formation of centrioles and cilia with symmetry (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 19.Rodrigues-Martins A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). SAS-6 is one of the conserved centriole which is a component of the cartwheel (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 18.Kilburn C.L. Pearson C.G. Romijn E.P. Meehl J.B. Giddings Jr., T.H. Culver B.P. Yates 3rd, J.R. Winey M. J. Cell Biol. 2007; 178: 905-912Crossref PubMed Scopus (132) Google Scholar, B.P. Meehl J.B. Giddings Jr., T.H. Winey M. Biol. Cell. PubMed Scopus (20) Google Scholar). However, the of SAS-6 in and in cartwheel formation The study provides into the formation of a SAS-6 with a symmetry that could serve as a building for the cartwheel central remains unclear is an that the of procentriole assembly and SAS-6 tetramers to and are components of the signaling that centriole (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar, J. J. M. R. Nigg E.A. Dev. Cell. 2007; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). that the protein is a protein for centriole formation and functions early in the centriole process S. J. Y. A. A. J. T. 2008; PubMed Scopus Google Scholar). It would be to further the of SAS-6, and in the assembly of the central tubule and procentriole in Chlamydomonas (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar), Drosophila A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and B.P. Meehl J.B. Giddings Jr., T.H. Winey M. Biol. Cell. PubMed Scopus (20) Google Scholar), outer centriole components in the of SAS-6, and in Chlamydomonas and Drosophila the 9-fold symmetry is The that outer centriole as the of the cartwheel that the role of the cartwheel is to symmetry. It be that studies in C. elegans not a cartwheel with a central tubule. a central tube is to the microtubules (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar). In SAS-6 with S. Delattre M. Cerutti L. Baumer K. Gönczy P. Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (293) Google Scholar), a protein that is not conserved in organisms that centrioles. SAS-6 and are required for the formation of the central tube in C. elegans (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar). It is that the central tube in C. elegans is a of and SAS-6, which would for its studies be to by in vitro in C. elegans to 7.4 S tetrameric structures by SAS-6 in E. coli the of SAS-6 in a that may not the of the The high density structures from P. pastoris suggest that a eukaryotic the of SAS-6 in the of centriolar the presence of in Drosophila cells to further SAS-6 as by the of native 50 S SAS-6 from Drosophila for the stability of of the results of higher order structures from Drosophila cells SAS-6 are The of tetramers and structures subunit show that the 7.4 S structures are stable that remain after the of higher order structures the of SAS-6 tetramers to form higher order structures in vitro when with embryonic suggest that the SAS-6 tetramers serve as stable in higher order structures. This that in to SAS-6 tetramers, central tubule assembly also requires in that the of SAS-6 in the cell as tetramers and Fig. that tetramers from of SAS-6 structures and that tetramers into higher order structures suggest that SAS-6 tetramers and are required to into a higher order biochemical and in vitro studies be to the that are essential for the assembly and stability of the central it was proposed that of SAS-6 form the central tubule with a one subunit (17.Strnad P. Leidel S. Vinogradova T. Euteneuer U. Khodjakov A. Gönczy P. Dev. Cell. 2007; 13: 203-213Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). studies are required to studies show that SAS-6 forms stable tetramers, and the central tubule may be of tetramers. This be in the by in SAS-6 that the formation of tetramers and to in central tubule. These studies the of that are found at the SAS-6 tetramers be organized into the central tubule at in A ring of tetramers each is to the of the central tubule is one In the central tubule in the would be of subunits of the of the central tubule after studies would A ring of tetramers that the central tubule in a central tubule is This be into a 9-fold symmetry as of tetramers would be that of the central that each has a in would a of at the we that the central tubule has a that be to be and the of the central tubule is which is the required for a of A of tetramers is a a 9-fold central tubule one subunit is then the central tubule would be organized into tetramers that one to a studies are required to the internal of SAS-6 in the central was also proposed that SAS-6 the procentriole by and a structure that to of the which are as A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, A. Riparbelli M. Callaini G. Glover D.M. Bettencourt-Dias M. Cell Cycle. 2008; 7: 11-16Crossref PubMed Scopus (54) Google Scholar). It is that SAS-6 tetramers and assembly to higher order structures are the units the of has been EM was used to the centriole and multiple RNAi and studies a role for SAS-6 early in centriole formation (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 16.Leidel S. Delattre M. Cerutti L. Baumer K. Gönczy P. Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (293) Google Scholar, P. Leidel S. Vinogradova T. Euteneuer U. Khodjakov A. Gönczy P. Dev. Cell. 2007; 13: 203-213Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 19.Rodrigues-Martins A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar), but of the central tubule and its internal The results from the study suggest that SAS-6 self-assembles into tetramers that serve as building blocks assembly of a cartwheel central tubule. IntroductionCentrioles are conserved microtubule-based organelles with 9-fold symmetry organized around a central tubule and spoke structure termed the “cartwheel.” This conserved 9-fold symmetry is also shared with basal body and cilia (1.Azimzadeh J. Bornens M. Nigg E.A. Centrosomes in Development and Disease. Wiley-VCH, Weinheim, Germany2004: 93-116Google Scholar, 2.Dippell R.V. Proc. Natl. Acad. Sci. U.S.A. 1968; 61: 461-468Crossref PubMed Scopus (209) Google Scholar, 3.Vorobjev I.A. Chentsov Yu. S. J. Cell Biol. 1982; 93: 938-949Crossref PubMed Scopus (275) Google Scholar). Such architectural similarity is indicative of the role of the centriole as a template for cilia formation. Cilia are microtubule-rich cellular organelles involved in signaling pathways and sensory functions in eukaryotic cells (4.Rosenbaum J. Curr. Biol. 2002; 12: R125Abstract Full Text Full Text PDF PubMed Google Scholar, 5.Scholey J.M. Anderson K.V. Cell. 2006; 125: 439-442Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Centrioles also recruit a pericentriolar matrix to form centrosomes, major microtubule organizing centers required for cell proliferation and differentiation (6.Badano J.L. Teslovich T.M. Katsanis N. Nat. Rev. Genet. 2005; 6: 194-205Crossref PubMed Scopus (199) Google Scholar, 7.González C. Tavosanis G. Mollinari C. J. Cell Sci. 1998; 111: 2697-2706Crossref PubMed Google Scholar). In ciliated cells, one of the centrioles inherited by the daughter cell migrates to the plasma membrane after cell division to serve as a template for the cell cilium. However, when a centriole duplicates, the new centriole does not use the existing centriole as a template (8.Phillips D.M. J. Cell Biol. 1967; 33: 73-92Crossref PubMed Scopus (44) Google Scholar, 9.Rodrigues-Martins A. Riparbelli M. Callaini G. Glover D.M. Bettencourt-Dias M. Science. 2007; 316: 1046-1050Crossref PubMed Scopus (196) Google Scholar), raising the question of how the new centriole establishes its 9-fold symmetry.Centriole biogenesis is a multistep process that includes an intermediate known as the procentriole (10.Azimzadeh J. Bornens M. J. Cell Sci. 2007; 120: 2139-2142Crossref PubMed Scopus (168) Google Scholar). The procentriole has an internal cartwheel structure that is required for the stability of the centriole and the 9-fold symmetry of cilia (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The cartwheel has a central tubule from which nine spokes emanate, each attaching to centriolar microtubules (supplemental Fig. S1A). Based on the structure of the cartwheel, it is expected that the central tubule would itself display 9-fold symmetry (12.Strnad P. Gönczy P. Trends Cell Biol. 2008; 18: 389-396Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) (supplemental Fig. S1A). It has been proposed that the central tubule generates the 9-fold symmetry of the centriole (13.Cavalier-Smith T. J. Cell Sci. 1974; 16: 529-556Crossref PubMed Google Scholar, 14.Satir P. Satir B. J. Theor. Biol. 1964; 7: 123-128Crossref PubMed Scopus (11) Google Scholar). However, the composition of the central tubule and the mechanisms underlying its biogenesis remain unknown.SAS-6 is a coiled-coil protein that is required for centriole biogenesis (15.Dammermann A. Müller-Reichert T. Pelletier L. Habermann B. Desai A. Oegema K. Dev. Cell. 2004; 7: 815-829Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 16.Leidel S. Delattre M. Cerutti L. Baumer K. Gönczy P. Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (293) Google Scholar). Overexpression of SAS-6 produces multiple procentrioles (17.Strnad P. Leidel S. Vinogradova T. Euteneuer U. Khodjakov A. Gönczy P. Dev. Cell. 2007; 13: 203-213Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). In Chlamydomonas and Tetrahymena, SAS-6 localizes to the center of the cartwheel (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 18.Kilburn C.L. Pearson C.G. Romijn E.P. Meehl J.B. Giddings Jr., T.H. Culver B.P. Yates 3rd, J.R. Winey M. J. Cell Biol. 2007; 178: 905-912Crossref PubMed Scopus (132) Google Scholar). Null mutants of SAS-6 in Chlamydomonas (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and Drosophila (11.Nakazawa Y. Hiraki M. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 2169-2174Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 19.Rodrigues-Martins A. Bettencourt-Dias M. Riparbelli M. Ferreira C. Ferreira I. Callaini G. Glover D.M. Curr. Biol. 2007; 17: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) lack the cartwheel. The SAS-6 null mutants of Chlamydomonas and Drosophila are also defective in establishing the 9-fold symmetry of centrioles. In Caenorhabditis elegans, RNAi studies show that SAS-6 is required for the formation of a centriole central tube. Direct evidence that SAS-6 is required for central tube assembly in worms comes from time resolved electron tomography of C. elegans embryos demonstrating that centriole formation begins with assembly of a central tube, a process that requires SAS-6 (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar). This study also implied that the tube could itself organize 9-fold symmetry, but it was not clear if the process was driven by steric constraints or was patterned by intrinsic cues on the outer surface of the tube. It was proposed that SAS-6 brings together nine pre-centriolar units called enatosomes, which then form a tube-like centriole precursor (22.Rodrigues-Martins A. Riparbelli M. Callaini G. Glover D.M. Bettencourt-Dias M. Cell Cycle. 2008; 7: 11-16Crossref PubMed Scopus (54) Google Scholar) and that SAS-6 is the repeating subunit of the central tubule (12.Strnad P. Gönczy P. Trends Cell Biol. 2008; 18: 389-396Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar).To investigate the mechanism of SAS-6 function, we employed a Drosophila model that allows for biochemical isolation and electron microscopy (EM) 2The abbreviations used are: EMelectron microscopyPCMpericentriolar materialγ-TuRCγ-tubulin ring complexγ-TuSCγ-tubulin small complexesGFPgreen fluorescent proteinPipes1,4-piperazinediethanesulfonic acidPBSphosphate-buffered saline. imaging of centrioles and cartwheels. In vertebrates the cartwheel is transient and is restricted to the “procentriole” stage (23.Alvey P.L. Cell Biol. Int. Rep. 1986; 10: 589-598Crossref PubMed Scopus (20) Google Scholar). In worms the cartwheel is not apparent (21.Pelletier L. O'Toole E. Schwager A. Hyman A.A. Müller-Reichert T. Nature. 2006; 444: 619-623Crossref PubMed Scopus (292) Google Scholar), and in protozoa it is restricted to the proximal end of basal bodies (18.Kilburn C.L. Pearson C.G. Romijn E.P. Meehl J.B. Giddings Jr., T.H. Culver B.P. Yates 3rd, J.R. Winey M. J. Cell Biol. 2007; 178: 905-912Crossref PubMed Scopus (132) Google Scholar, 24.Hiraki M. Nakazawa Y. Kamiya R. Hirono M. Curr. Biol. 2007; 17: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Drosophila centrioles are structurally similarly to the procentrioles of higher organisms (7.González C. Tavosanis G. Mollinari C. J. Cell Sci. 1998; 111: 2697-2706Crossref PubMed Google Scholar), and cartwheels are constitutive components of Drosophila centrioles. EM studies of in situ centrioles in Drosophila embryos reported that the cartwheel extends through the full length of the centriole (25.Callaini G. Whitfield W.G. Riparbelli M.G. Exp. Cell Res. 1997; 234: 183-190Crossref PubMed Scopus (66) Google Scholar).Here, we report that in Drosophila, the central tubule of the cartwheel has repeating subunits and that SAS-6 localizes at the central tubule. Recombinant SAS-6 produced in Escherichia coli or Pichia pastoris forms tetrameric structures that are fractionated at 7.4 S. Most of the native SAS-6 exists as 7.4 S structures, which is the most common soluble form of SAS-6. Native SAS-6 is also found as part of 50 S structures and centrosomes. Elevated levels of SAS-6 in Drosophila cells resulted in higher order structures built from multiple central tubule-like structures. Affinity purification of these high density SAS-6 complexes contained tetramers. Upon disassembly, these higher order complexes resulted in tetramers. In vitro assembly of these tetramers could then be assembled into high density complexes in the presence of embryonic extract. Collectively these data suggest that the SAS-6 tetramers serve as building blocks of a higher order central tubule at the core of centriole